Cells were grown in 24-well plates with 0.170mm glass bottom (In Vitro Scientific). Where indicated, the cells were pre-extracted before fixation with pre-extraction buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 1.5 mM MgCl2, 300 mM sucrose, 0.5% Triton-X 100, 1mM DTT, 5 μg/ml leupeptin, 2 μg/ml aprotinin, 0.1 mM PMSF) for 20 minutes at 4°C, washed by PBS and then immediately fixed with 4% formaldehyde for 15 minutes at room temperature. Cells were stained with primary antibodies: anti-ubiquitylated conjugateed mouse FK2 antibody (1:500; Enzo, cat. n.: BML-PW8810), anti-VCP (1:500; Abcam; cat. n.: ab11433), anti-NPL4 (1:500; Novus Bio, cat. n.: NBP1-82166), HSP70 (1:100; Enzo, cat. n.: ADI-SPA-830), HSF1 (1:500; Cell Signaling, cat. n.: 4356) anti-ubiquitin lys48-specific (1:500; Merck Millipore, clone Apu2), Sumo2/3 (1:500; Abcam, cat. n.: ab3742), TDP-43 (1:300; Proteintech, cat. n.: 10782-2-AP) and appropriate Alexa Fluor 488 and 568 secondary antibodies (Invitrogen, 1:1000). Cytochrome c was stained by Alexa Fluor 555 conjugated mouse anti-cytochrome c antibody according manufacture’s protocol (BD Pharmingen, cat. n.: 558700).
Anti vcp
Manufactured by Abcam
Anti-VCP is a primary antibody that recognizes the Valosin Containing Protein (VCP), also known as p97. VCP is a highly conserved and abundant ATPase involved in various cellular processes. This antibody can be used to detect and study the VCP protein.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 and 568 secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Anti npl4, by Novus Biologicals (2 mentions)
Anti ubiquitylated conjugateed mouse fk2 antibody, by Enzo Life Sciences (2 mentions)
Clone apu2, by Abcam (2 mentions)
Alexa fluor 555 conjugated mouse anti cytochrome c antibody, by BD (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti v5, by Thermo Fisher Scientific (1 mentions)
Er stress antibody sampler kit, by Cell Signaling Technology (1 mentions)
Anti tubulin, by Santa Cruz Biotechnology (1 mentions)
Ab11433, by Abcam (1 mentions)
Amylo glo, by Biosensis (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti acly, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti hspa5, by Abcam (1 mentions)
Anti cd44, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti pstat3 tyr705, by Cell Signaling Technology (1 mentions)
6 protocols using anti vcp
1
Immunohistochemistry of Cellular Stress Markers
2
Subcellular Fractionation and Protein Detection
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Cells were collected in 1% NP-40 containing 0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl2. After centrifugation at 15,000 × g for 5 min, the supernatant was collected and analyzed as the soluble fraction. The pellet was re-suspended in 1% SDS containing 0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl2 and analyzed as the insoluble fraction. Those fractions were boiled for 5 min in SDS–PAGE sample buffer containing 0.125 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 10% 2-mercaptoethanol, and 0.004% bromophenol blue and loaded onto a 5–20% or 10–20% polyacrylamide gradient gel. The ER stress antibody sampler kit was obtained from Cell Signaling Technology. Blue native-PAGE was performed as previously described (Bin et al, 2011 (link)). Anti-V5 (Invitrogen), anti-tubulin (Santa Cruz), anti-ubiquitinated proteins (Biomol), anti-FLAG (Sigma), and anti-VCP (Abcam) antibodies, and an anti-ER stress antibody sampler kit (Cell Signaling) were used for protein detection.
3
Immunohistochemistry of Cellular Stress Markers
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Cells were grown in 24-well plates with 0.170mm glass bottom (In Vitro Scientific). Where indicated, the cells were pre-extracted before fixation with pre-extraction buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 1.5 mM MgCl2, 300 mM sucrose, 0.5% Triton-X 100, 1mM DTT, 5 μg/ml leupeptin, 2 μg/ml aprotinin, 0.1 mM PMSF) for 20 minutes at 4°C, washed by PBS and then immediately fixed with 4% formaldehyde for 15 minutes at room temperature. Cells were stained with primary antibodies: anti-ubiquitylated conjugateed mouse FK2 antibody (1:500; Enzo, cat. n.: BML-PW8810), anti-VCP (1:500; Abcam; cat. n.: ab11433), anti-NPL4 (1:500; Novus Bio, cat. n.: NBP1-82166), HSP70 (1:100; Enzo, cat. n.: ADI-SPA-830), HSF1 (1:500; Cell Signaling, cat. n.: 4356) anti-ubiquitin lys48-specific (1:500; Merck Millipore, clone Apu2), Sumo2/3 (1:500; Abcam, cat. n.: ab3742), TDP-43 (1:300; Proteintech, cat. n.: 10782-2-AP) and appropriate Alexa Fluor 488 and 568 secondary antibodies (Invitrogen, 1:1000). Cytochrome c was stained by Alexa Fluor 555 conjugated mouse anti-cytochrome c antibody according manufacture’s protocol (BD Pharmingen, cat. n.: 558700).
4
Immunostaining of Ubiquitin Modifications
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Cells were fixed 5 min after microthermal damage with 4% formaldehyde for 15 min at room temperature, washed with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 5 min. After PBS washes, the cells on the plastic inserts were immunostained with primary antibody for 1 h at room temperature (anti-K48-ubiquitin, Apu2, Merck Millipore; anti-K63-ubiquitin, Apu3, Merck Millipore; anti-VCP, Abcam, ab11433), followed by PBS washes and staining with Alexa Fluor 568-conjugated secondary antibody for 60 min at room temperature. Nuclei were visualized by DAPI staining at room temperature for 2 min. For the beta-aggregates visualization, NIAD-4 (Sigma) dye was directly added to culture media (300 nM), and aggregates were detected in live cells after laser irradiation. AmyloGlo staining was performed according to the manufacturer’s instruction (Biosensis). Briefly, cells were irradiated by the 561 nm laser, fixed with formaldehyde for 15 min at room temperature, washed with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 5 min and washed. Next, the slide was incubated with 70% ethanol for 5 min, washed with distilled water, and stained with 1X AmyloGlo reagent for 15 min, followed by quick washes in 0.9% saline and distilled water.
5
Antibodies for Western Blot Analysis
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The antibodies used in this study were as follows: anti-FBXO43 (Sigma, HPA024292, 1:500), anti-GAPDH (Santa-Cruz, sc-32233, 1:2,000), anti-Flag (Sigma, F1804, 1:2,000), anti-β-actin (Santa Cruz, sc-69879, 1:2,000), anti-VCP (Abcam, ab109240, 1:10,000), anti-CD44 (Abcam, ab51037, 1:5,000), anti-PCNA (CST, #2586, 1:1,000), anti-ACLY (Abcam, ab40793, 1:1,000), anti-HSPA5 (Abcam, ab108615, 1:1,000), anti-ACTN4 (Abcam, ab108198, 1:1,000), anti-rabbit IgG (Santa-Cruz, sc-2004, 1:2,000), anti-mouse IgG (Santa-Cruz, sc-2005, 1:2,000), anti-rabbit IgG (CST, #7074), and anti-mouse IgG (CST, #7076, 1:10,000).
6
Western Blot Protein Analysis
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For Western blot analysis, cells were lysed with the Cell Lysis Buffer (Cell Signaling, 9803S). Subsequentially, samples were run on a SDS-PAGE and blotted onto PVDF membranes (Merck Chemicals, ISEQ00010) overnight. After blocking, primary antibodies were incubated overnight in 5% milk-TBST using the following concentrations: anti-HAS2 (1:500, Abcam, ab140671), anti-VCP (1:5000, Abcam, ab109240), anti-ACTB (1:5000, Sigma Aldrich, A5441), anti-LRRC15 (1:1000, Abcam, ab150376), anti-E-Cadherin (1:2000, BD Biosciences, 610181), anti-HAPLN1 (1:1000, Bio-Techne, AF2608), anti-pP65 (Ser536) (Cell Signaling, #3033), anti-pFAK (Tyr925) (Cell Signaling, #3284), anti-pSTAT3 (Tyr705) (Cell Signaling, #9145). Secondary antibodies goat anti-rabbit HRP, rabbit anti-mouse HRP (1:2500, DAKO, P0448, P0260) or donkey anti-goat HRP (1:2500, R&D, HAF109) were incubated for 1 h prior to detection with SuperSignal West Pico Plus Chemisubstrate (VWR International, PIER34577).
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