Calnexin

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany

Calnexin is a type I membrane protein that functions as a molecular chaperone in the endoplasmic reticulum (ER). It plays a key role in the quality control of newly synthesized glycoproteins, assisting in their proper folding and preventing the release of misfolded proteins. Calnexin interacts with monoglucosylated glycoproteins, retaining them in the ER until they achieve their native conformation.

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Lab products found in correlation

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Spelling variants of this product

Anti-calnexin (45 citations) Rabbit polyclonal anti-calnexin (8 citations) Rabbit anti-calnexin (6 citations) Rabbit anti-Calnexin antibody (3 citations) Mouse anti-Calnexin (3 citations) Calnexin (H-70), (3 citations) Goat anti-mouse calnexin antibody (2 citations) Calnexin antibody (2 citations) Goat polyclonal anti-calnexin (2 citations) Goat anti-Hsp60 (N-20) and rabbit anti-Calnexin (H-70) (2 citations)

88 protocols using calnexin

Immunofluorescence Imaging of RVFV Proteins

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HSAECs were seeded on poly-L-lysine coated glass coverslips (Neuvitro, Cat# GG-22-1,5-PDL) in 6-well plates at 5 × 10⁵ cells/well seeding density. Cells were infected (MOI 1) with MP12, V5Gn¹⁰⁵ or V5Gn²²⁹ viruses. At 24 hpi, cells were fixed with 4% paraformaldehyde and permeabilized as previously described (Pinkham et al., 2016 (link)). Cells were then probed for RVFV Gn (clone 4D4, 1:5000 BEI Resources), RVFV Gc (clone 4B6, 1:5000, BEI Resources, Cat# NR-43184), TGN46 (AbD Serotec, Cat# AHP500G), and calnexin (Santa Cruz, Cat# sc11397) expression. The following secondary antibodies (1:500) were used to visualize the primary antibodies, Alexa Fluor 488 anti-mouse (Thermo Fisher Scientific, Cat# A-11001), Alexa Fluor 568 anti-rabbit (Thermo Fisher Scientific, Cat# A-11011), and Alexa Fluor 568 anti-sheep (Thermo Fisher Scientific, Cat# A-21099). Cells were counterstained with DAPI before mounting coverslips on glass slides. Slides were imaged using the Nikon Eclipse TE 2000-U microscope with a 60x oil immersion lens. Five images were taken per sample, with one representative shown.

Bracci N., de la Fuente C., Saleem S., Pinkham C., Narayanan A., García-Sastre A., Balaraman V., Richt J.A., Wilson W, & Kehn-Hall K. (2022). Rift Valley fever virus Gn V5-epitope tagged virus enables identification of UBR4 as a Gn interacting protein that facilitates Rift Valley fever virus production. Virology, 567, 65-76.

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Extracellular Vesicle Protein Analysis

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Briefly, a 20 μg EV sample was dissolved in sample buffer, sonicated and boiled at 95 °C for 5 min, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-Protein II electrophoresis cells (Bio-Rad, USA), as described earlier (24 (link), 26 (link)). The proteins were visualized using the Coomassie brilliant blue. For western blotting, separated protein bands were transferred (25 V, 150 min) into a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, USA). The membrane was blocked with 5% BSA for an hour, then washed and incubated overnight at 4 °C with a primary antibody. Primary antibodies include Calnexin (1:500, Santa Cruz, USA), CD63 (1:500, Santa Cruz, USA), CD9 (1:500, Santa Cruz, USA), and TSG 101 (1:500, Santa Cruz, USA). Then, the blot was washed and treated with a secondary anti-mouse (1:50000, Sigma-Aldrich, Germany) for two hours. The band conjugates were detected by enhanced chemiluminescence (ECL) detection reagent (Amersham, GE Healthcare, Buckinghamshire, UK) visualized by the Uvitec documentation system (UK).

Davari A., Hajjaran H., Khamesipour A., Mohebali M., Mehryab F., Shahsavari S, & Shekari F. (2023). Amphotericin B-Loaded Extracellular Vesicles Derived from Leishmania major Enhancing Cutaneous Leishmaniasis Treatment through In Vitro and In Vivo Studies. Iranian Journal of Parasitology, 18(4), 514-525.

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Quantitative Western Blot Analysis of Extracellular Vesicles

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Cells and sEV were lysed with Pierce™ RIPA lysis buffer (Pierce Protein Biology/Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor (Sigma‐Aldrich) and protein content determined using the BCA protein assay (Pierce Protein Biology/Thermo Fisher Scientific). Polyacrylamide gel electrophoresis (PAGE) was performed using standard reagents from Invitrogen/Thermo Fisher Scientific. Samples were denatured for 5 min at 95°C, loaded onto Bolt™ 4–12% Bis‐Tris Plus Gels, run at 200 V for 28 min and transferred to a nitrocellulose membrane at 10 V for 60 min. Membranes were blocked for 1 h at room temperature using Intercept^® (TBS) blocking buffer (LI‐COR, Lincoln, NE, USA) and subsequently probed with primary antibodies overnight at 4°C, including CD63 (Mouse monoclonal IgG1, Clone Ts63; Invitrogen/Thermo Fisher Scientific), CD9 (Mouse monoclonal IgG1, Clone Ts9; Invitrogen/Thermo Fisher Scientific), CD81 (Mouse IgG1, Clone M38; Invitrogen/Thermo Fisher Scientific) and Calnexin (Mouse monoclonal IgG1, Clone AF18; Santa Cruz, Dallas, TX, USA). Proteins were visualized with IRDye 800CW goat anti‐mouse (LI‐COR) using the Odyssey CLX (LI‐COR). Quantitative analysis of protein intensities was performed using Image Studio 5.2 software (LI‐COR).

Wang X., Wilkinson R., Kildey K., Ungerer J.P., Hill M.M., Shah A.K., Mohamed A., Dutt M., Molendijk J., Healy H, & Kassianos A.J. (2021). Molecular and functional profiling of apical versus basolateral small extracellular vesicles derived from primary human proximal tubular epithelial cells under inflammatory conditions. Journal of Extracellular Vesicles, 10(4), e12064.

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Western Blot Antibody Protocol

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The antibodies used for Western blotting in this study were: hTERT (1531-1, clone Y182, Epitomics), SK2 (ab37977, Abcam), MKRN1 (ab72054, Abcam), rabbit V5 (ab9116, Abcam), Ubiquitin (3933S, Cell Signaling Technology), HA (3724, Cell Signaling Technology), Calnexin (Sc6465, Santa Cruz Biotechnology), Lamin B (Sc6216, Santa Cruz Biotechnology), mouse V5 (R96025, Invitrogen, USA).

Selvam S.P., De Palma R.M., Oaks J.J., Oleinik N., Peterson Y.K., Stahelin R.V., Skordalakes E., Ponnusamy S., Garrett-Mayer E., Smith C.D, & Ogretmen B. (2015). Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation. Science signaling, 8(381), ra58.

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Immunoblotting of HDAC Isoforms

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Immunoblotting was performed as previously described(16 (link)). Briefly, pediatric human and neonatal rat RV homogenates were prepared and concentrations quantified as above for HDAC catalytic activity assay. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes (BioRad) and probed with antibodies for HDAC1 (Cell Signaling Technology, 5356), HDAC2 (Cell Signaling Technology, 5113), HDAC3 (Cell Signaling Technology, 3949), HDAC4 (Cell Signaling Technology, 5392), HDAC5 (Cell Signaling Technology, 2082), HDAC6 (Santa Cruz Biotechnology, 11420), HDAC7 (Cell Signaling Technology, 2882), calnexin (Santa Cruz Biotechnology, 11397).

Blakeslee W.W., Demos-Davies K.M., Lemon D.D., Lutter K.M., Cavasin M.A., Payne S., Nunley K., Long C.S., McKinsey T.A, & Miyamoto S.D. (2017). Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy. Pediatric research, 82(4), 642-649.

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Characterization of Vaginal Fluid Exosomes

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Vaginal fluid exosomes (VFE, isolated as described above) were analyzed for exosomal surface markers by FACS analysis utilizing an Exo-Flow kit from SBI (cat # EXOFLOW150A-1). Following the manufacturer’s instructions, biotinylated capture antibodies (CD9 or CD63, provided with kit, the biotinylated CD81 antibody was not available from the manufacturer) were conjugated to Exo-Flow FACS magnetic streptavidin 9.1 µm beads to allow for the efficient capture of exosomes expressing these surface markers. Then 70 µg exosomes were captured, stained with FITC, and subjected to FACS analysis. As a control, beads were treated with all reagents in the kit, but instead of exosomes, plain PBS was added.
We also performed western blotting to detect the presence of exosomal surface markers. Semen exosome samples were used as a control. Exosome samples were lysed and ca 20 µg of each sample was run on SDS-PAGE, transferred and blotted with exosome marker antibodies CD63 (SBI, Palo Alto, CA, cat # EXOAB-CD63A-1) and CD81 (SBI, Palo Alto, CA, cat # EXOAB-CD81A-1). As a negative and a purity control, filters were blotted with the ER-derived marker calnexin (Santa Cruz Biotechnology, Santa Cruz, CA, cat # sc-11397).

Smith J.A, & Daniel R. (2016). Human vaginal fluid contains exosomes that have an inhibitory effect on an early step of the HIV-1 life cycle. AIDS (London, England), 30(17), 2611-2616.

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Protein and miRNA Extraction and Analysis

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Protein extraction and Western blot analysis were carried out as described [17 (link)]. Details of the antibody dilutions used for Western blot are as follows: LXRα (1:500 dilution, #ab41902, Abcam, Cambridge, UK), ABCA1 (1:1000, #NB400-105, Novus Biologicals, Cambridge, UK), GAPDH (1:1000, #sc-365062, Santa Cruz Biotechnology, TX, USA), and Calnexin (1:1000, #sc-11397, Santa Cruz Biotechnology, TX, USA).
RNA extraction and quantitative real time PCR are described in detail in the Supplementary material. In short, total RNA was extracted with Trifast (Peqlab, Erlangen, Germany) and reverse transcribed (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Carlsbad, CA). The quantity of RNA used in quantitative real time PCR was optimized prior to obtain ct values within the range of 18–30.
For mature miR-206 quantification the miRCURY LNA™ Universal RT microRNA PCR system (Exiqon, Vedbaek, Denmark) was used together with miR-206 or U6 (reference gene) specific predesigned LNA primers. Quantitative real time PCR was carried out using gene specific primers and QuantiFast SYBR Green PCR Kit (Qiagen, Germany) on LC480 (Roche Diagnostics, Basel, Switzerland). All samples were normalized to cyclophilin A mRNA expression. Experiments were carried out in triplicates.

Vinod M., Chennamsetty I., Colin S., Belloy L., De Paoli F., Schaider H., Graier W.F., Frank S., Kratky D., Staels B., Chinetti-Gbaguidi G, & Kostner G.M. (2014). miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages. Biochimica et Biophysica Acta, 1841(6), 827-835.

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EV Protein Expression Profile Analysis

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Cells or EVs were lysed with RIPA buffer (Cell Signalling Technology Cat. No. 9806) and sonication. Then, 10 µg of total protein of each sample was separated on 10% SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane (GE Healthcare, Freiburg, Germany). The membranes were blocked overnight with 5% milk at 4°C and incubated with primary antibodies (ABs) against CD63 (1:1000, BioLegend Cat. No. 353013), TSG101 (1:1000, Abcam Cat. No. ab83), HSP70 (1:1000, Enzo Life Sciences Cat. No. ADI-SPA-810-F,) and Calnexin (1:1000, Santa Cruz Biotechnology Cat. No. sc-23954) for 2 h at RT. After washing, the blots were incubated with HRP-conjugated anti-mouse IgG secondary antibody (1:3000, Cell Signalling Technology Cat. No. 7076) for 1 h at RT. Protein expression was visualised using the Immobilon Forte Western HRP Substrate (Merck Millipore, USA) and Bio-Rad ChemiDoc MP imaging system (Munich, Germany).

Bachurski D., Schuldner M., Nguyen P.H., Malz A., Reiners K.S., Grenzi P.C., Babatz F., Schauss A.C., Hansen H.P., Hallek M, & Pogge von Strandmann E. (2019). Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView. Journal of Extracellular Vesicles, 8(1), 1596016.

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Western Blot Analysis of Stress Proteins

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Western blotting analysis was performed as described previously [46 (link)] using primary antibodies against LCN2 (R&D Systems), ERp57, ERAP1 (Abcam; Cambridge, MA, USA), ERO1α (Novus Biologicals; Littleton, CO, USA), HO-1 (Enzo Life Sciences; Farmingdale, NY, USA), CRT, NRF2, calnexin, p47phox, β-actin (Santa Cruz Biotechnology; Dallas, TX, USA), and PDI (Cell Signaling; Danvers, MA, USA). Goat anti-rabbit-IgG-HRP (Cell Signaling), donkey anti-goat IgG (Santa Cruz Biotechnology), and rabbit anti-mouse-IgG-HRP (Calbiochem, San Diego, CA, USA) were used as secondary antibodies. The blots were quantified using an Alliance Mini 4 M (UVItec, Cambridge, UK). Western blot bands corresponding to each protein were quantified, and the intensity of each target protein was normalized to the intensity of the β-actin loading control. The normalized ratio of the control was set as 1.0 to compare target protein abundance in different sample. The normalized ratios of target proteins were used to compare target protein abundance in different samples. The normalized ratio is shown at the bottom of the blots. The normalized intensity values of three different experiments are plotted as mean ± standard deviation (SD).

Choi J.A., Cho S.N., Lee J., Son S.H., Nguyen D.T., Lee S.A, & Song C.H. (2021). Lipocalin 2 regulates expression of MHC class I molecules in Mycobacterium tuberculosis-infected dendritic cells via ROS production. Cell & Bioscience, 11, 175.

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Immunofluorescence and Western Blot Analysis

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The following antibodies were used for immunofluorescence, filter retardation and/or western blot: WO1 amyloid-specific antibody (mouse monoclonal) (O’Nuallain & Wetzel, 2002 (link)), beta tubulin (mouse monoclonal, TUB 2.1, Sigma-Aldrich), FUS/TLS (rabbit monoclonal, EPR5812, Abcam), hsc70 (rabbit monoclonal, EP1531Y, Abcam), lamin B (goat polyclonal, C-20, Santa Cruz Biotechnology, Inc.), lamin A/C (mouse monoclonal, Santa Cruz Biotechnology Inc.), nucleolin C23 (mouse monoclonal, MS-3, Santa Cruz Biotechnology Inc.), nucleophosmin B23 (rabbit polyclonal, H-106, Santa Cruz Biotechnology Inc.), U1-70K/SmB/B’ (human serum, ASR53, von Mikecz serum database), ubiquitin (rabbit polyclonal, Sigma-Aldrich), polyQ (mouse monoclonal, 5TF1-1C2 MAB1574, Millipore), pan hnRNP (mouse monoclonal, C-6, Santa Cruz Biotechnology Inc.), calnexin (rabbit polyclonal, Santa Cruz Biotechnology Inc.), 20S proteasome alpha subunits (mouse monoclonal, MCP231, Millipore), 20S proteasome (rabbit polyclonal, kind gift of B Dahlmann), CaMKII alpha (mouse monoclonal, Abcam), CaMKII alpha phospho T286 (rabbit polyclonal, Abcam).

Arnhold F., Gührs K.H, & von Mikecz A. (2015). Amyloid domains in the cell nucleus controlled by nucleoskeletal protein lamin B1 reveal a new pathway of mercury neurotoxicity. PeerJ, 3, e754.

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