Cells at a concentration of 1 × 105 cells/mL were plated into a 6-well dish. After 3 days of incubation, cells in each well were lysed with lysis buffer at 4 °C for 30 min. The lysates were loaded and analyzed using SDS-PAGE following standard protocols. Primary antibodies used were anti-REG4 antibody (1:500, Abcam, Cambridge, UK) and anti-Actin antibody (1:1000, Proteintech, Wuhan, China). Secondary antibodies used were anti-mouse antibodies (1:1000, Proteintech, Wuhan, China) and anti-rabbit antibodies (1:1000, Proteintech, Wuhan, China).
Anti actin antibody
Manufactured by Proteintech
Sourced in United States
Anti-actin antibody is a laboratory reagent used to detect and quantify the presence of actin, a key structural protein found in eukaryotic cells. It can be used in various applications such as Western blotting, immunocytochemistry, and immunohistochemistry to visualize and analyze actin distribution and expression levels.
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Lab products found in correlation
Anti flag antibody, by Merck Group (3 mentions)
Anti mouse antibodies, by Proteintech (1 mentions)
Anti rabbit antibodies, by Proteintech (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Caspase 9, by Proteintech (1 mentions)
Cyt c, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit antibody, by Proteintech (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
Caspase 3, by Proteintech (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Anti gst, by Proteintech (1 mentions)
Tg and t cho detection kit, by Nanjing Jiancheng (1 mentions)
9 protocols using anti actin antibody
1
REG4 Protein Expression Analysis
2
Western Blotting Analysis of HIV Protein Expression
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Western blotting was performed as previously described.53 (link) In briefly, 36 h post infection of HEK293T cells with the HIVNL4-3-ΔE-YFP virus, which had been transfected with hCas13a/crRNA plasmid, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 10 mM Tris [pH 7.5], 1 mM EDTA). Equal amounts of cell lysates were separated in SDS-12% PAGE (WB1103, Beijing Biotides Biotechnology). Proteins were transferred onto nitrocellulose membranes (Whatmann). The membranes were probed with anti-CA-p24 antibody (Sino Biological, 11695-V08E), anti-FLAG antibody (Sigma, F3165), anti-actin antibody (Proteintech, 60008-1-Ig), or anti-GFP antibody (Proteintech, 66002-1-Ig), followed by incubation with IRDye secondary antibodies (1:20,000). Protein bands were visualized on a Li-Cor Odyssey instrument.
3
Protein Extraction and Western Blot
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Total proteins were extracted from 10-day-old seedlings grown on ½ MS plates using 5% SDS and boiled for 10 min at 95°C before running on SDS–polyacrylamide gel electrophoresis gel. FLAG-tagged proteins were detected with horseradish peroxidase–conjugated anti-FLAG antibody (Sigma-Aldrich, A8592-1MG). Actin protein detected by an anti-actin antibody (Proteintech, 60008-1-Ig) was used as a loading control. All Western blots were developed using the ECL Plus Western Blotting Detection System (GE Healthcare, RPN2132) and chemiluminescent imaging using an ImageQuant LAS 4000 (GE Healthcare).
4
Ischemic Brain Injury Protein Analysis
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After 24 h of reperfusion, the mice (n = 6, for each group) were decapitated and ischemic brains were collected. Protein lysates of ischemic hemispheres in each group were subjected to 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with blocking solution (5% skim milk in TBST) and incubated overnight at 4°C with the primary rabbit monoclonal antibodies respectively (Bax, 1:1000; Bcl-2, 1:500; Caspase-3, 1:1000; Caspase-9, 1:500; Proteintech Group, U.S.A.; CytC, 1:1000; Abcam, U.K. and PARP-1, 1:500; Cell Signal, U.S.A.). Secondary incubations were performed with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:3000; Proteintech Group, U.S.A.). An anti-actin antibody (1:1000; Proteintech Group, U.S.A.) was used as loading controls. Immunoreactive proteins were visualized using enhanced chemiluminescence (ECL) and the signals were quantified by densitometry with a Western blotting detection system (Quantity One, Bio-Rad, U.S.A.).
5
Virus Infection and Cell Culture Protocols
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Human muscular rhabdomyosarcoma (RD) cells and human embryonic kidney 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS supplemented with L-glutamine, penicillin and streptomycin (Gibco BRL, Grand Island, NY, USA).
EV71 is a Fuyang strain (GenBank accession no. FJ439769.1). To conduct virus infections, cells were infected with EV71 at different MOIs (multiplicity of infection). Unbound viruses were washed off 2 h after infection. The enterovirus 68 (EV68) strain that was used in this study is a Beijing strain (GenBank accession no. KF726085). The reverse genetic system was utilized to produce influenza virus A/WSN/33 (H1N1)57 (link).
Amphotericin B was purchased from Sigma–Aldrich. Mouse anti-VP1 antibody was purchased from Abnova, mouse anti-EV71 antibody from Millipore, anti-Actin antibody from Proteintech, anti-SCARB2 antibody from R&D systems, IRD Fluor 800-labeled IgG secondary antibodies from Li-Cor Inc., Lincoln, NE. The anti-EV68 3C antibody was obtained as previously described58 (link).
EV71 is a Fuyang strain (GenBank accession no. FJ439769.1). To conduct virus infections, cells were infected with EV71 at different MOIs (multiplicity of infection). Unbound viruses were washed off 2 h after infection. The enterovirus 68 (EV68) strain that was used in this study is a Beijing strain (GenBank accession no. KF726085). The reverse genetic system was utilized to produce influenza virus A/WSN/33 (H1N1)57 (link).
Amphotericin B was purchased from Sigma–Aldrich. Mouse anti-VP1 antibody was purchased from Abnova, mouse anti-EV71 antibody from Millipore, anti-Actin antibody from Proteintech, anti-SCARB2 antibody from R&D systems, IRD Fluor 800-labeled IgG secondary antibodies from Li-Cor Inc., Lincoln, NE. The anti-EV68 3C antibody was obtained as previously described58 (link).
6
Antibody and Reagent Utilization Protocol
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Antibodies and reagents used in the study are as follows: anti‐TSP50 antibody was prepared and purified by Abcam (1:1000). Anti‐G6PD (25413‐1‐AP, 1:1000), anti‐GST (10000‐0‐AP, 1:1000), anti‐Flag (20543‐1‐AP, 1:1000), anti‐SIRT2 (19655‐1‐AP, 1:1000), anti‐pan acetylation antibody (66289‐1‐Ig, 1:1000), anti‐O‐glycosylation antibody (20415‐1‐AP, 1:1000), rabbit IgG, anti‐actin antibody (60008‐1‐Ig, 1:5000) and mouse IgG were purchased from Protein‐tech Group. Anti‐G6PD K171ac was designed and prepared by Absin Bioscience Inc (1:500), and anti‐KAT9 was purchased from Bioss Antibodies (bs‐20539R, 1:500). NADP+ was purchased from MedChemExpress. NADP+/NADPH detection kit and G6PD activity detection kit were purchased from Beyotime. TG and T‐CHO detection kit were purchased from Nanjing Jiancheng Bioengineering Institute. A glutaraldehyde solution was purchased from Sigma‐Aldrich.
7
Western Blot Analysis of XBP-1 Splicing
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Whole cell protein extracts were prepared from EndoCβH2-Cre cells using radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing a cocktail of protease and phosphatase inhibitors (Roche Applied Science). The extracted proteins (15 ng) were resolved on a 4–20% Mini-PROTEAN (Bio-Rad Laboratories) electrophoresis gel and immunoblotted with anti–spliced XBP-1 and anti-actin antibodies (Proteintech and MilliporeSigma, respectively) (Supplementary Table 1 ). The densitometry of the protein bands was calculated with ImageJ software.
8
Investigating CEP85L Translation Initiation
9
Culturing and Antibody Analysis of H1299 Lung Cancer Cell Line
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