Confocal microscopy was performed as described previously22 (link) using the following primary antibodies: β-tubulin (IgG1, clone D66; Sigma-Aldrich), Ki67 (ab15580; Abcam), LAMP-1 (Abcam), SNX1 (BD Biosciences), EEA1 (BD Biosciences), FAM21 (Millipore), calreticulin (Abcam), and LC3 (L8918; Sigma-Aldrich). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 0.1 mg/ml) where indicated. For labeling of acidic organelles, LysoSensor Green DND-189 (1 μM) was added to cells in growth medium and incubated at 37°C for 30 min before imaging. A Zeiss LSM710 confocal microscope with a 63× 1.4 NA Plan-Apochromatic oil differential interference contrast objective was used, and image acquisition was performed using LSM710 version 3.2SP2 software. Images were analyzed with ImageJ (NIH) and edited using Adobe Photoshop 7.0 and Illustrator CS2 software.
Calreticulin
Manufactured by Abcam
Sourced in United Kingdom, United States
Calreticulin is a calcium-binding protein that plays a role in the regulation of calcium homeostasis. It is found in the lumen of the endoplasmic reticulum (ER) and participates in the folding and quality control of newly synthesized proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (9 mentions)
β actin, by Abcam (7 mentions)
Gapdh, by Abcam (3 mentions)
Tom20, by Santa Cruz Biotechnology (3 mentions)
α tubulin, by Merck Group (3 mentions)
Lamp1, by Abcam (2 mentions)
Lamin a c, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Histone h3, by Abcam (2 mentions)
Hmgb1, by Abcam (2 mentions)
Eif2α, by Cell Signaling Technology (2 mentions)
Tsg101, by Abcam (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Illustrator cs2, by Adobe (1 mentions)
Ki67 ab15580, by Abcam (1 mentions)
Lc3 l8918, by Merck Group (1 mentions)
34 protocols using calreticulin
1
Confocal Microscopy of Organelle Markers
2
Comprehensive EV Protein Profiling Protocol
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Cells and EVs were lysed with working solution of the RIPA buffer (Beyotime) containing a protease inhibitor cocktail (Roche). Proteins were separated by sodium dodecyl sulfate–12% polyacrylamide gel (SDS–PAGE) electrophoresis and blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were incubated with PBST (PBS and 0.1% Tween 20) containing 5% skim milk for 1 h, and successively incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Membranes were developed using an enhanced chemoluminescence (ECL) system (Clinx Science Instruments, Shanghai, China). The primary and secondary antibodies included β-actin (1:5000, Genetex), CD63, CD9, Alix, voltage-dependent anion-selective channel (VDAC) 137 (link), Calreticulin, Lamin A/C, Tau, Map-2 (1:1000, Abcam, Cambridge, UK), Cleaved caspase-3 (aCasp3), Cav-1 (1:1000, CST, Boston, MA), HRP-conjugated goat anti-rabbit IgG (Genshare, Xian, China) and HRP-conjugated goat anti-mouse IgG (HRP) (Genshare).
3
Western Blot Analysis of Cell Signaling Proteins
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WB analysis was performed as previously described.51 (link) Cells were lysed using the M-PER Mammalian Protein Extraction Reagent (ThERMO Scientific), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. The following antibodies were used: HMGB1 (1:1000, ab79823, Abcam), HSP70 (1:1000, ab181606, Abcam), Calreticulin (1:1000, ab92516, Abcam), RAGE (1:1000, ab216329, Abcam), phosphorylated NF-κB [1:1000, 3033, Cell Signaling Technology (CST)], NF-κB (1:1000, 8242, CST), phosphorylated Akt (1:1000, 4060S, CST), Akt (1:1000, 4691S, CST), phospho-MAPK Family Antibody Kit (1:1000, 9910, CST), a MAPK Family Antibody Kit (1:1000, 9926, CST), β-Actin (1:1000, 4970S, CST) and GAPDH (1:1000, 60004-1-Ig; Proteintech). Proteins were visualized by using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd).Images were prepared using ChemiDoc XRS + system (BioRad, China) and quantification analyses were performed by Bio-Rad Image Lab software (BioRad, China).
4
Immunofluorescence Staining of Subcellular Structures
5
Protein Extraction and Western Blot Analysis
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After 48 hours of transfection, cells were lysed in RIPA buffer containing a protease inhibitor cocktail (P2714; Sigma), centrifuged for 2 minutes at 10,000 × g. Denaturing of the protein lysate was done by adding 1X Laemmli buffer containing 5% β-mercaptoethanol and incubation at 99 °C for 10 minutes. Proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane using a semidry transfer apparatus (Bio-Rad). The membranes were incubated in blocking buffer (LI-COR Biosciences) for 1hr and incubated with primary polyclonal antibody against ANO5 (1:1000; Abcam) and calreticulin (1:5000; Abcam) in blocking buffer containing 0.1% Tween20 at 4 °C overnight. The blots were stripped and reprobed with monoclonal antibodies against β-actin (1:5000; Sigma) as an internal control. HRP-conjugated secondary mouse antibody (Amersham) at a dilution of 1:3000 and the ECL detection system (Amersham) was used.
6
Western Blot Protein Analysis
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Cells were washed and lysed in RIPA protein lysis buffer containing a complete protease inhibitor cocktail (Roche). Proteins were then separated using SDS-polyacryamide gel electrophoresis, transferred to nitrocellulose membranes and incubated with the indicated primary antibodies: DCK (Abcam ab186128), acetylated histone H3 (Cell Signalling #4353), histone H3 (Cell Signalling #4499), calreticulin (Abcam ab2907) and β-actin (Abcam ab8229).
7
Immunoblotting Analysis of Cellular Proteins
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Whole cell protein lysates were boiled after diluted using 5 × SDS loading buffer and subjected to different densities (8–15%) of sodium dodecyl sulfate (SDS), then transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with blocking buffer (Beyotime Biotechnology, cat. no. P0023B) for 1 h and incubated with the following primary antibodies overnight at 4 °C: cGAS (cat.no: ab224144, Abcam), STING (cat.no.13647S, CST), Calreticulin (CRT, cat.no: ab92516, Abcam), HMGB1 (cat.no: ab79823, Abcam), γ-H2A.X(S139) (cat.no:ab81299, Abcam) and β-actin (cat. no. AC026, Abclonal), β-actin was set as loading control. The membranes were washed thrice for 5 min in a mixture of PBS and Tween-20 (PBST), incubated with anti-rabbit IgG, HRP-linked second antibody secondary antibodies (cat. no. 7074; CST) for 2 h, and washed again thrice for 5 min in PBST. Immunoblotting bands were observed using ChemiScope Series 6000Touch(Clinx Science Instruments Co.,Ltd) after incubated with Pierce™ ECL Plus western blotting substrate (cat. no. 32132; Life).
8
Western Blot Analysis of Signaling Proteins
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This was performed as described56 (link),57 (link). The following primary antibodies were used: EPHB4 (D1C7N) (1: 1000, Cell Signaling #14960), EPHB4 (1:500, Abcam #ab73259), phospho-eiF2α S51 (1:1000, Cell Signaling #9721 S), eiF2α (1:000, Cell Signaling #9722), phospho-IRE1(S724) (1:1000, Abcam #ab48187) , c-Myc (Y69) (1:5000, Abcam #ab32072), GLUT3 (1:5000, Abcam #ab191071), Calreticulin (1:400, Abcam #ab2907), HMGB1 (Abcam #ab18256), Actin (1:10000, Cell Signaling #5125), GAPDH (1:5000, Cell Signaling #3683), Phospho-Src Family Tyr416 (1:1000, Cell Signaling #2101), Phospho-p38 MAPK Thr180/Tyr182 (1:1000, Cell Signaling #9211), and Phospho-4EBP1 Thr37/46 (236B4) (1:1000, Cell Signaling #2855). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse (1:5000, Biorad) or goat anti-rat (1:5000, Santa Cruz) for 1 h, the binding of secondary antibodies were detected with the Supersignal West Femto Maximum Sensitivity Substrate detection system (Pierce) followed by visualization. Quantification analyses were performed by Biorad ChemiDoc Imager and Bio-Rad Image Lab software.
9
Comprehensive Immunohistochemistry Panel
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Primary antibodies and their respective species and dilutions used in this study are as follows: AIF1 (Cell Signaling Technology 5318; rabbit, 1:200), aSMA/FITC conjugate (Abcam F3777; mouse, 1:200), calreticulin (Abcam ab92516; rabbit, 1:200), CD42b (Abcam ab227669; rabbit, 1:200), collagen IV (Abcam ab6586; rabbit, 1:200), cytokeratin 8 (R&D Systems MAB3165; mouse IgG1, 1:200), endomucin (Santa Cruz Biotechnology sc-65495; rat IgG2a, 1:200), GFAP (Thermo Fisher Scientific 14-9892-82; mouse IgG1, 1:200), MIB1 (Abcam ab124929; rabbit, 1:200), nephrin (Progen GP-N2; guinea pig, 1:100), synaptopodin (Synaptic Systems 163 004; guinea pig, 1:100), β-amyloid (Thermo Fisher Scientific 715800; rabbit, 1:200), vimentin (Progen GP53; guinea pig, 1:200), vWF (Agilent A008229-2; rabbit, 1:200), laminin (Abcam ab11575; rabbit, 1:200) and CD144 VE-CAD (Thermo Fisher Scientific 14-1441-82; rat, 1:200).
10
Immunofluorescence Microscopy of Transfected Cells
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Cells were seeded over glass coverslip in 24-wells plates and plasmid transfected the day after with Lipofectamine-2000. The following day cells were fixed with fresh 4% PFA pH8.0, permeabilized and blocked with PBS containing 0.1% Triton X-100 and 10% heat-inactivated normal goat serum (PBSTH). Primary antibodies were incubated overnight at 4 °C at dilutions recommended by the providers in PBSTH. After washing in PBS, secondary fluorescently tagged antibodies were incubated for 1 h at room temperature in PBSTH. For non-permeabilized immunofluorescence with anti-HA antibodies the protocol of Briley et al. [100 (link)] was followed with the exception of the replacement of BSA for 10% heat-inactivated goat serum. The efficiencies of transfection and the total numbers of HA-positive cells in each experiment were determined per condition. Images were taken with an LSM700 confocal microscope (0.4 µm slices) at the University of Geneva’s Imaging Facility and processed using FIJI software. Primary antibodies used were anti-: HA-Tag (Cell Signaling 2367; 1/100), TGN46 (ABCAM ab50595, 1/200), cMYC (Santa Cruz Biotechnology sc-40; 1/100), CALRETICULIN (ABCAM ab2907;1/75), EEA1 (Cell Signaling No. 3288; 1/100), and LAMP1 (Cell Signaling No. 9091; 1/200).
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