Calnexin

Manufactured by Enzo Life Sciences

Sourced in United States, United Kingdom

Calnexin is a calcium-binding chaperone protein that is primarily located in the endoplasmic reticulum (ER) of eukaryotic cells. It plays a key role in the quality control of protein folding and assembly within the ER.

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Lab products found in correlation

Pvdf membrane, by Merck Group (5 mentions) Actin, by Merck Group (5 mentions) Sox10, by R&D Systems (4 mentions) Cy3 donkey anti rabbit igg h l, by Jackson ImmunoResearch (3 mentions) Gapdh, by Merck Group (3 mentions) β actin, by Merck Group (3 mentions) Lamp1, by Merck Group (2 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Nitrocellulose membrane, by GE Healthcare (2 mentions) Ps473 akt, by Cell Signaling Technology (2 mentions) Bradford assay, by Bio-Rad (2 mentions) Talin, by Abcam (2 mentions) Gm130, by BD (2 mentions) P75ntr, by Merck Group (2 mentions) Neurofilament, by Abcam (2 mentions) Donkey anti goat igg h l alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions) Serine 63 phosphorylated c jun, by Cell Signaling Technology (2 mentions) C jun, by Cell Signaling Technology (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) β tubulin, by Merck Group (2 mentions)

38 protocols using calnexin

Immunofluorescence and Western Blot Assays for Neuronal Markers

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Immunofluorescence: SOX10 (R&D Systems, 1:100, AF2864, RRID:AB_442208) donkey anti-goat IgG (H + L) Alexa Fluor 488 (Invitrogen, 1:1000, A11057), Anti-Beta III Tubulin (Sigma Aldrich, 1:1000, AB9354, RRID:AB_570918), NF200 (Abcam, 1:1000, ab72997, RRID:AB_1267598), SARM1 rabbit polyclonal antibodies (kind gift from Professor Hsueh, 1:500) and purchased from Abcam (Ab226930, 1:2000, RRID:AB_2893433), tdTomato (SICGEN, 1:500, RRID:AB_2722750) donkey anti-goat IgG (H + L) Alexa Fluor 488 (Invitrogen, 1:1000, A11057), Cy3 donkey anti-rabbit IgG (H + L) (Jackson Immunoresearch, 1:500, 711-165-152), Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Seco Alexa Fluor 488 (Invitrogen, 1:1000, A-21202), Donkey anti-Chicken IgY, Alexa Fluor 647 (Merck, 1:1000,15389818), DAPI (Thermo scientific, 1:2000, 62248).
Western blot: CALNEXIN (Enzo Life Sciences, 1:1000, ADI-SPA-860-D, RRID:AB_312058), JUN (Cell Signalling Technology, 1:1000, 9165, RRID:AB_2130165), EGR2 (EMD Millipore, 1:500, ABE1374, RRID:AB_2715555), MPZ (Aves Labs, 1:2000, PZO, RRID:AB_2313561), MBP (EMD Millipore, 1:1000, AB9348, RRID:AB_2140366), Anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, 1:2000, 7076S), Anti-rabbit HRP-linked antibody (Cell Signaling Technology, 1:2000, 7074S), Goat pAb to Chicken IgY H + L (HRP) (Abcam, 1:2000, ab97135).

Fazal S.V., Mutschler C., Chen C.Z., Turmaine M., Chen C.Y., Hsueh Y.P., Ibañez-Grau A., Loreto A., Casillas-Bajo A., Cabedo H., Franklin R.J., Barker R.A., Monk K.R., Steventon B.J., Coleman M.P., Gomez-Sanchez J.A, & Arthur-Farraj P. (2023). SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice. Frontiers in Cellular Neuroscience, 17, 1158388.

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Immunofluorescence Localization of Lipid Regulators

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HepG2 cells were fixed by 4% paraformaldehyde for 10 minutes at room temperature after being washed by PBS twice. Then cells were blocked and permeabilized with 0.1% Triton X-100 (MP Biomedicals) and 10% FBS diluted in PBS for 30 minutes at room temperature. After that, cells were incubated with primary antibody SREBP1 (Thermo Fisher Scientific, 1:100), SREBP2 (Abcam, 1:100), INSIG1 (Proteintech, 1:200), CALNEXIN (Enzo, 1:200), and PDI (MilliporeSigma, 1:200) incubated at 4°C overnight. After 5 washes with PBS, cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 568 goat anti-mouse IgG (H+L) secondary antibody (Life Technologies) for 1 hour at room temperature with gentle shaking. At last, the samples were washed with PBS 3 times before staining the nucleus with DAPI for 5 minutes. Immunofluorescence images were obtained and analyzed with Zeiss 710 NLO confocal microscopy.
For fluorescence intensity quantification, ImageJ was applied, and relative intensity was quantified by intensity divided by view area. For colocalization, the rate was quantified using ImageJ with colocalization finder. For each sample, no less than 5 representative images were analyzed to quantify the fluorescence intensity values and calculate an average. Experiments were repeated 3 times individually at least.

Xu Y., Tao J., Yu X., Wu Y., Chen Y., You K., Zhang J., Getachew A., Pan T., Zhuang Y., Yuan F., Yang F., Lin X, & Li Y.X. (2021). Hypomorphic ASGR1 modulates lipid homeostasis via INSIG1-mediated SREBP signaling suppression. JCI Insight, 6(19), e147038.

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Immunofluorescence and Western Blot Protocols

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Immunofluorescence antibodies: JUN (Cell Signaling Technology, rabbit 1:800), Ki67 (Abcam, rabbit 1:100), L1 (Chemicon International, rat 1:50), MCAM (Origene, rabbit 1:200), MPZ (AvesLab, chicken 1:1000), NGFR (Thermo Fisher Scientific, mouse 1:100), SOX10 (R and D Systems, goat 1:100), donkey anti-goat IgG (H + L) Alexa Fluor 555 conjugate (Molecular Probes, 1:1000), donkey anti-rabbit IgG (H + L) Alexa Fluor 488 conjugate (Molecular Probes, 1:1000), donkey anti-chicken IgG (H + L) Alexa Fluor 488 conjugate (Jackson ImmunoResearch Labs, 1:1000), goat anti-rat IgG (H + L) Alexa Fluor 555 conjugate (Molecular Probes, 1:1000), Cy3 donkey anti-mouse IgG (H + L) (Jackson Immunoresearch, 1:500), and Cy3 donkey anti-rabbit IgG (H + L) (Jackson Immunoresearch, 1:500).
Antibodies used for Western blotting: CALNEXIN (Enzo Life Sciences, rabbit 1:1000), JUN (Cell Signaling Technology, rabbit 1:1000), GAPDH (Sigma-Aldrich, rabbit 1:5000), HDAC5 (Santa Cruz, mouse 1:500), KROX-20 (Millipore, rabbit 1:500), MCAM (Origene, rabbit 1:1000), MPZ (AvesLab, chicken 1:1000), NGFR (Covance, rabbit 1:1000), TYRP1 (Sigma-Aldrich, rabbit 1:1000), IgY anti-chicken HRP-linked (Sigma-Aldrich, 1:2000), IgG anti-mouse and IgG anti-rabbit HRP-linked (Cell Signaling Technology, 1:2000). A list of the antibodies used can be found online (Key Resources Table).

Velasco-Aviles S., Patel N., Casillas-Bajo A., Frutos-Rincón L., Velasco E., Gallar J., Arthur-Farraj P., Gomez-Sanchez J.A, & Cabedo H. (2022). A genetic compensatory mechanism regulated by Jun and Mef2d modulates the expression of distinct class IIa Hdacs to ensure peripheral nerve myelination and repair. eLife, 11, e72917.

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Quantifying Protein Expression in Brain Regions

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The procedure of Western blotting has been previously described (Sun et al., 2009 (link)). Immediately after cocaine prime-induced restatement, rats were rapidly decapitated and brains were removed, flash frozen, and stored in −80°C until used. Brain regions were dissected, including PFC, NAc, caudate-putamen (CPu), bed nucleus of the stria terminalis (BNST), hypothalamus, amygdala (Amy), dorsal hippocampus (dHip), ventral hippocampus, and ventral tegmental area. Protein was extracted by sonication and loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels then transferred to polyvinylidene difluoride membranes. Membranes were then incubated with primary antibody against p-ERK (1:5000), ERK (1:2000) (both from Cell Signaling, Beverly, MA), p-GluA1 Ser845 (1:1000; Millipore, Billerica, MA), GluA1 (1:10000; Abcam, Cambridge, MA), OTR (1:500; Abbiotec, San Diego, CA), and calnexin (1:10000; Enzo Life Sciences, Farmingdale, NY), followed by their appropriate secondary antibodies. Antibody binding was detected by using an enhanced chemiluminescence kit (ECL Plus; GE Healthcare Bio-Sciences, Piscataway, NJ). The integrated density of protein band was measured using ImageJ software (National Institute of Health, Bethesda, MD).

Zhou L., Sun W.L., Young A.B., Lee K., McGinty J.F, & See R.E. (2014). Oxytocin Reduces Cocaine Seeking and Reverses Chronic Cocaine-Induced Changes in Glutamate Receptor Function. International Journal of Neuropsychopharmacology, 18(1), pyu009.

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Immunological Analysis of ALS Biomarkers

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Antibodies against TDP‐43 (Sigma‐Aldrich Cat# SAB4200006, Cosmo Bio Co Cat# CAC‐TIP‐TD‐P09), GFP (UC Davis/NIH NeuroMab Facility Cat# 73‐132), β‐actin (Sigma‐Aldrich Cat# A5316), calnexin (Enzo Life Sciences Cat# ADI‐SPA‐860‐F), VPS4B (Sigma‐Aldrich Cat# PA5‐30316 for IHC and Proteintech Group Cat# 17673‐1‐AP for immunoblotting and immunofluorescence), ErbB4 (Santa Cruz Biotechnology Cat# sc‐283), phosphor‐ErbB4‐Y1056 (Santa Cruz Biotechnology Cat# sc‐33040), FGFR1 (Novus Cat# NB600‐1287), phosphor‐FGFR1‐Y654 (Abcam Cat# ab59194), myc‐tag (Santa Cruz Biotechnology Cat# sc‐40), HA‐tag (Sigma‐Aldrich Cat# H9658 and Roche Cat# 3F10), anti‐mouse and rabbit HRP‐coupled secondary antibodies and anti‐mouse and rabbit Alexa‐coupled secondary antibodies (Life Technologies) are commercially available. TO‐PRO 3 and transferrin‐Alexa 555 were purchased from Life Technologies, and DAPI and EZ‐Link sulfo‐NHS‐LC biotin from Sigma‐Aldrich. β2‐Transferrin protein levels from undiluted CSF samples derived from sporadic ALS patients and controls were quantified using a commercially available ELISA kit (www.mybiosource.com, MBS923983) following the manufacturer's instructions. Recombinant NRG1 and FGF1 were purchased from R&D Systems.

Schwenk B.M., Hartmann H., Serdaroglu A., Schludi M.H., Hornburg D., Meissner F., Orozco D., Colombo A., Tahirovic S., Michaelsen M., Schreiber F., Haupt S., Peitz M., Brüstle O., Küpper C., Klopstock T., Otto M., Ludolph A.C., Arzberger T., Kuhn P, & Edbauer D. (2016). TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons. The EMBO Journal, 35(21), 2350-2370.

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Antibody Reagents for Cellular Signaling

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The following antibodies were purchased: Kit (M-14), STAT5 (C-17), Erk2 (K-23), Src (Src2) and from Santa Cruz Biotechnology; Kit[pTyr719], Kit[pTyr703], Akt (40D4), Akt[pT308] (C31E5E), STAT5[pTyr694] (D47E7), PDGFRα (D13C6), Erk[pThr202/pTyr204] (E10), golgin97 (D8P2K) and Src[pY416] (D49G4) from Cell Signaling Technology (Danvers, MA, USA); p85, Kit[pTyr568/570], pTyr (4G10) and Src (327) from Millipore; golgin97 (CDF4) and calnexin (AF18) from Thermo Scientific Pierce (Rockford, IL, USA); calnexin from Enzo (Farmingdale, NY, USA); GM130 (35), Yes (1) and Fyn (25) from BD Transduction Laboratories (Franklin Lakes, NJ, USA); LAMP1 from Sigma and Kit (104D2) from Biolegend (San Diego, CA, USA). Horseradish peroxidase-labeled secondary antibodies were purchased from the Jackson Laboratory (Bar Harbor, MA, USA). Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA).

Obata Y., Horikawa K., Takahashi T., Akieda Y., Tsujimoto M., Fletcher J.A., Esumi H., Nishida T, & Abe R. (2017). Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors. Oncogene, 36(26), 3661-3672.

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Molecular Characterization of TDP-43 Pathology

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TDP‐43 (Cosmo Bio Co, TIP‐TD‐P09), TDP‐43 (Proteintech, 10782‐2‐AP), TDP‐43 phospho‐S409/410 (Cosmo Bio Co., Ltd, TIP‐PTD‐P02), TDP‐43 (C‐terminal; Proteintech, 12892‐1‐AP), ChAT (Merck, AB144P), GFP (UC Davis/NIH NeuroMab Facility, N86/8 and N86/38), PSMC4 (Bethyl Laboratories, A303‐850A and A303‐849A), tagRFP (Thermo Fisher Scientific, R10367), KPNA1 clone 114‐E12 (Thermo Fisher Scientific, 37‐0800), calnexin (Enzo Life Sciences, ADI‐SPA‐860‐F), HA 3F10 (Merck, 11867423001), GA 5F2 (Mackenzie et al, 2013), control IgG from mouse serum (Merck, I5381), and HCS CellMask™ Deep Red Stain (Thermo Fisher Scientific, H32721) were used.

Khosravi B., LaClair K.D., Riemenschneider H., Zhou Q., Frottin F., Mareljic N., Czuppa M., Farny D., Hartmann H., Michaelsen M., Arzberger T., Hartl F.U., Hipp M.S, & Edbauer D. (2020). Cell‐to‐cell transmission of C9orf72 poly‐(Gly‐Ala) triggers key features of ALS/FTD. The EMBO Journal, 39(8), e102811.

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Western Blot Analysis of Cellular Proteins

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Equal aliquots from each fraction separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 1 hr in 5% milk/Tris-buffered saline and probed overnight at 4°C with the following primary antibodies diluted in 5% milk/Tris-buffered saline containing 0.1% Tween-20: calnexin (Enzo Life Sciences, Farmingdale, NY, USA), syntaxin1a (Abcam, Cambridge, MA, USA), mGluR5, PKCε, phospho-(Ser729)-PKCε (all from Millipore, Billerica, MA, USA) and ERK1/2 and phospho-ERK1/2 (both from Cell Signaling Technology, Danvers, MA, USA). After incubation with an appropriate horseradish peroxidase-conjugated secondary antiserum (Jackson ImmunoResearch, West Grove, PA, USA), immunoreactive bands on the membranes were detected by ECL+ chemiluminescence reagents on an X-ray film (GE Healthcare, Piscataway, NJ, USA). Subsequently, blots were stripped and re-probed with calnexin and syntaxin-1a antibodies to monitor biotinylation of intracellular proteins and to normalize for unequal loading and/or transfer of proteins. The integrated band density of each protein sample was measured using NIH Image J software version 1.32j (RRID: SCR_003070). All primary antibodies used in this study are described in detail in Table 1.

Schwendt M, & Olive M.F. (2016). Protein kinase Cε activity regulates mGluR5 surface expression in the rat nucleus accumbens. Journal of neuroscience research, 95(4), 1079-1090.

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Immunoblotting Analysis of Antiviral Signaling

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Cells were harvested, washed with PBS, and lysed on ice for 30 minutes in lysis buffer (1% CHAPS (Pierce) in PBS containing a protease inhibitor cocktail tablet (Roche)). After centrifugation at 800g for 10 minutes at 4°C to remove debris, lysates were sparated by SDS-PAGE and transferred onto PVDF membranes (Millipore). The membranes were probed using antibodies against viperin (MaP.VIP) [8 (link)], or commercial antibodies to MAVS (Abcam and Santa Cruz), MDA-5 (Cell Signaling Technology), RIG-I (Cell Signaling Technology), calnexin (Enzo), Grp94 (Enzo), FACL4 (Abcam), TFPβ (LifeSpan BioSciences), Tim23 (BD Biosciences), and non-phosphorylated IRF3 and phosphorylated IRF3 (Cell Signaling Technology). Secondary antibodies conjugated to Horse Radish Peroxidase were purchased from Jackson ImmunoResearch. Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Hee J.S, & Cresswell P. (2017). Viperin interaction with mitochondrial antiviral signaling protein (MAVS) limits viperin-mediated inhibition of the interferon response in macrophages. PLoS ONE, 12(2), e0172236.

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Epithelial-Mesenchymal Transition Markers Assay

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Hygromycin, puromycin, zeocin, collagen, fibronectin, and laminin substrates were purchased from Life Technologies. Matrigel was obtained from Corning. Transwell inserts (CLS3422) were purchased from Sigma. Antibodies used in our studies were: Vimentin (1:4000 WB; 1:500 IF; Abcam), E-Cadherin (1:250 IF in 2D culture; 1:50 IF in 3D culture; BD Biosciences), Snail (1:500 WB; Cell Signaling), Slug (1:500 WB; Cell Signaling), Calnexin (1:1000 WB; Enzo), Bim (1:250 WB; Enzo), laminin V (1:50 IF in 3D culture; EMD Millipore), Claudin1 (1:500 WB; Invitrogen), Twist1 (1:500 WB; Santa Cruz), PCNA (1:250 WB; Santa Cruz), CyclinD (1:250 WB; Santa Cruz), and VRK1 (1:1000 WB; Sigma).

Mon A.M., MacKinnon AC J.r, & Traktman P. (2018). Overexpression of the VRK1 kinase, which is associated with breast cancer, induces a mesenchymal to epithelial transition in mammary epithelial cells. PLoS ONE, 13(9), e0203397.

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