Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors in ice for 30 min followed by centrifugation at 12,000 × g for 15 min at 4°C. Immunoblotting was performed using the anti-cleaved PARP mAb (1:1,000), anti-Bax mAb (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-cytochrome c mAb (1:500), anti-caspase-3 mAb (1:1,000; Abcam, Cambridge, MA, USA), anti-HBc mAb (1:500; Milipore, Billerica, MA, USA), anti-β-actin and anti-secondary antibodies were purchased from ZSGB-BIO (Beijing, China).
Anti β actin
Manufactured by ZSGB-BIO
Sourced in China, United States, United Kingdom
Anti-β-actin is a primary antibody used in western blot analysis to detect the presence and quantity of the β-actin protein. β-actin is a widely expressed cytoskeletal protein that is often used as a loading control or reference protein in protein expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (9 mentions)
Anti flag, by Merck Group (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Ripa buffer, by Beyotime (4 mentions)
Bca protein assay kit, by Boster Bio (3 mentions)
Anti bcl 2, by Cell Signaling Technology (3 mentions)
Anti gapdh, by ZSGB-BIO (3 mentions)
Goat anti rabbit igg, by ZSGB-BIO (3 mentions)
Goat anti mouse igg, by ZSGB-BIO (3 mentions)
Bca kit, by Beyotime (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Anti stat1, by Cell Signaling Technology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti claudin 1, by Abcam (2 mentions)
Anti occludin, by Santa Cruz Biotechnology (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (2 mentions)
62 protocols using anti β actin
1
Apoptosis Signaling Pathway Analysis
2
Western Blot Analysis of Protein Expression
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The total protein was extracted and subjected to electrophoresis. Cell lysates were prepared by using RIPA protein extraction reagent (Beyotime Biotechnology) and protein concentrations were determined by Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Samples containing equal amounts of protein were separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Amersham Hybond). Membranes were blocked by 5% BSA in TBS/0.1% Tween‐20, then incubated overnight at 4°C. The expression level of each protein was detected by an enhanced chemiluminescence system (Vazyme Biotech). The primary Abs anti‐PD‐L1, anti‐UCHL1, anti‐STAT1, anti‐phospho‐STAT1, anti‐ERK1/2, anti‐phospho‐ERK1/2, anti‐P65, and anti‐phospho‐P65 were purchased from Cell Signaling Technology with catalog numbers E1L3N, D3T2E, 7649P, 9175S,4695P, 4370S, 3033T, and 8242T, respectively. The primary Abs anti‐AKT and anti‐phospho‐AKT were purchased from Epitomics with catalog numbers 1085‐S and 2214‐S, respectively. The primary Abs also included anti‐Flag (Cat# F1804; Sigma‐Aldrich) and anti‐β‐actin (Cat# PR‐0255; ZSGB‐BIO). The secondary Abs were goat anti‐rabbit IgG Ab or goat anti‐mouse IgG Ab (Cat# ZB‐2301 or Cat# ZB‐2305; ZSGB‐Bio).
3
Western Blot Analysis of Alzheimer's Markers
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Western blot analysis was performed as previously described [24 (link)]. Hippocampal tissue samples were homogenized in an ice-cold RIPA buffer (Beyotime, Jiangsu, China) supplemented with a protease inhibitor. The homogenates were centrifuged at 12,000 × g for 5 min at 4°C, supernatants were collected and their protein concentrations determined with the BCA protein assay kit (Beyotime, Jiangsu, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 8% or 10% polyacrylamide gels, depending on the molecular weight separation range needed. After bands were transferred to polyvinylidene difluoride (PVDF) membranes, the membranes were incubated at 4°C for overnight with the appropriate primary antibody, anti-BACE-1 (CST, Danvers, USA), anti-APP (CST, Danvers, USA) or anti-Aβ42 (Abcam, Cambridge, UK), each diluted 1:500. To detect labeled protein bands, the membranes were next incubated for 2 h with the appropriate fluorescently labeled secondary antibody at room temperature. Image-Pro Plus 6.0 software ( Media Cybernetics, Washington, USA) was used to analyze the fluorescence data for each blot. Anti-β-actin (1:750; ZSGB-BIO, Beijing, China) was also used as a protein loading control for each sample. Results are presented as the ratio of the intensity of the APP, BACE-1 and Aβ42 band to that of the β-actin band.
4
Protein Expression Analysis via Western Blot
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Each cell group was collected and lysed in RIPA buffer (Biosharp, HeFei, China) to complete protein extraction. The collected protein samples were added to a sodium dodecyl sulfate polyacrylamide gel (Activagen Bio, Hefei, China) at a ratio of 1:4 to separate the proteins. Then, the gels were electrotransferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked using 5% skim milk powder. Then, the following primary antibodies were added overnight at 4 °C: anti-β-Actin (Zs-BIO, USA, 1:1,000); anti-Pink1 (Affinity, USA, 1:1,000); anti-Parkin (Affinity, USA, 1:1,000); anti-Rab7 (ZENBIO, ChengDu, China, 1:1,000); anti-NLRP3 (Affinity, USA, 1:1,000); anti-Caspase-1 ( Affinity, USA, 1:1,000); anti-Podxl (ABclonal, WuHan, China, 1:1,000); anti-Beclin1 (Affinity, USA, 1:500); anti-LC3 II/LC3 I (Affinity, USA, 1:500); and anti-P62 (CST, USA, 1:1000). The membranes were washed with PBS buffer and incubated with the secondary antibody for 2 h at room temperature, after which the membrane signal was observed via an enhanced chemiluminescence kit (Thermo, USA). Film strips were analyzed using ImageJ software 1.8.0.
5
Metformin's Molecular Mechanisms Investigated
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Metformin was purchased from Melone Pharmaceutical (Dalian, China). Anti-GRP94 and anti-C/EBP β antibodies were obtained from Sangon Biotech (Shanghai, China). Anti-CHOP and anti-Caspase-3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-phosphorylated (p)-AMPKα was from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin was obtained from ZSGB-BIO (Beijing, China).
6
Tight Junction Protein Expression
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The duodenum was homogenized by using a bead mill (Biospec, USA) in RIPA buffer containing 1 mM PMSF (Solarbio, China). The BCA assay was used to measure the protein concentration. Equal amount of total proteins (80 μg/lane) was loaded on SDS-PAGE gel and transferred to PVDF membrane after separation. The membrane was then incubated with the following primary antibodies: anti-occludin (1:400, Santa Cruz Biotechnology), anti-claudin-1(1:2000, Abcam), and anti-β-actin (1:1500, ZSGB-BIO) at 4 °C overnight. After three 10-min washing with PBS (supplemented with 0.1% Tween 20), the membrane was incubated with appropriate secondary antibodies: HRP-conjugated goat anti-mouse, rabbit anti-goat or goat anti-rabbit (both 1:5000, ZSGB-BIO) antibody. The protein bands on the membrane were visualized following incubation with enhanced chemiluminescence substrate and the protein levels were quantified with Image J software.
7
Western Blot Analysis of Protein Markers
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Total proteins were isolated from the treated EC-1 cells using RIPA buffer (Solarbio) and stored at -80°C, according to the manufacturer’s instructions. Protein concentration was determined by measuring the absorbance (optical density, OD562), using BCA Protein Assay Kit (Biotech Well, Shanghai, People’s Republic of China). Briefly, 40 µg of total protein isolated from each sample was separated on 10%–15% sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAGE) and then transferred to polyvinylidene difluoride membrane (Millipore) at 100 mV for 1.5–2 h. The blots were blocked and incubated with primary antibodies overnight at 4°C, using anti-MMP-9 (1:200; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-vimentin (1:400), and anti-β-actin (1:1,000; ZsBio, Beijing, People’s Republic of China) antibodies. After washing thoroughly (3×10 min) with Tris-buffered saline–Tween 20 (TBST), the samples were incubated with goat anti-mouse secondary immunoglobulin G (IgG) (1:2,000; Bioss, Shanghai, People’s Republic of China) or goat anti-rabbit secondary IgG (1:2,000; Bioss) for 2 h at room temperature. The bands were detected using enhanced chemiluminescence method (BeyoECL Plus). All experiments were repeated three times.
8
Quantitative Analysis of Spinal Cord Proteins
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While under deep anesthesia (5% sevoflurane), the L4-L5 segments of spinal cord were isolated quickly and stored in -80 ºC. Tissue samples were homogenized in lysis buffer and centrifuged at 13,000 rpm for 10 min at 4 °C. Supernatant was removed. Bradford method was used to determine the protein concentration. Samples (70 μg) were separated on SDS-PAGE (6%) and transferred onto a nitrocellulose membrane. The filter membranes were blocked with 5% nonfat milk for 1 h at RT (room temperature) and incubated with the primary anti NR2B (rabbit affinity purified polyclonal antibody; 1:1000, Abcam, Hong Kong, China), anti REST (rabbit affinity purified polyclonal antibody; 1:300, Santa Cruz Biotechnology, Santa Cruz, CA) or anti β-actin (mouse affinity purified monoclonal antibody; 1:1000, ZSGB-BIO, Beijing, China). The membrane was washed with TBST buffer and incubated for 1h with the secondary antibody conjugated with horseradish peroxidase (1:5000) for 1 h at RT and visualized in ECL solution followed by film exposure. The density of specific bands was quantified with a computer-assisted imaging analysis system (IPLab software, Scanalytics, Fairfax, VA).
9
Investigating P2Y2 and Wnt Signaling
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ATP and iCRT‐14 were purchased from Sigma (St Louis, MO, USA). ATP was dissolved in sterile normal saline and used at a concentration of 100 μM. The iCRT‐14 was dissolved in DMSO and used at a concentration of 25 μM. The antibodies used in this study are as follows: anti‐β actin (TA‐09, ZSGB‐BIO, Beijing, China), anti‐P2Y2 (H‐70; SC‐20124, Santa Cruz, CA, USA), anti‐β‐catenin (61053, BD Biosciences, San Jose, CA, USA), anti‐Myc (K422; BS2462, Bioword Technology, St Louis, MN, USA), anti‐Axin2 (E2A6978, Enogene, Nanjing, China), anti‐CD44 (ZM‐0537, ZSGB‐BIO, Beijing, China), Ki‐67 (GM001, Gene Tech, Shanghai, China).
10
Western Blot Analysis of EMT Markers
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The Western bolt analysis was performed as described previously [18 (link)]. The protein concentrations of cell or tissue lysates were measured using the Bicinchoninic Acid Assay (BCA) (TIANGEN, Beijing, China, PA115). Afterward, 60 µg protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk for two hours at room temperature, immunoblotted with specific primary antibodies. The antibodies used in this experiment were as follows: anti-E-cadherin (CST, Boston, MA, USA, 3195S; 1:1, 000); anti-N-cadherin (CST, Boston, MA, USA, 13116S, 1:1, 000); anti-Vimentin (Abcam, Cambridge, UK, ab8978, 1:1, 000); anti-Slug (Abcam, Cambridge, UK, ab51772, 1:1, 000); anti-β-actin (ZSGB-BIO, Beijing, China; TA-09, 1:1, 000); anti-GAPDH (Santa Cruz, CA, USA, sc-25778, 1:1, 000); anti-MZF1 (Santa Cruz, CA, USA, 293218, 1:1, 000). All antibodies were used following the manufacturer’s instructions. Protein expression was detected using a chemiluminescence agent (Thermo, Waltham, MA, USA). ImageJ software (Bethesda, MD, USA) was employed to quantify the results.
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