HUVECs were lysed with a modified RIPA buffer (Beyotime Institute of Biotechnology, China) containing 1 mM phenylmethanesulfonyl fiuoride (PMSF). Membrane and cytosol fraction isolation was performed according to kit instructions (Proteintech, China). Protein content was determined using a Bradford assay normalized against bovine serum albumin (Sigma, USA). Protein samples were separated by SDS-PAGE gel and then electrotransferred to PVDF membranes (Millipore, USA). After incubation in blocking solution (5% non-fat dried milk, Aspen, USA), membranes were incubated over night at 4 °C with a primary antibody against β-Actin (Abbkine, Redlands, CA, USA), LC3, p62, IR, p-Akt, mTOR, p-mTOR (Cell Signaling Technology, Beverly, MA, USA), GSK3β, p-GSK3β, LDLR, Akt (Proteintech, China) used at 1:1000 dilution or GLUT1 (Cell Signaling Technology, Beverly, MA, USA) used at 1:500 dilution. Membranes were rinsed and incubated with goat anti-rabbit or goat anti-mouse secondary antibody (1:10000, Abbkine, Redlands, CA, USA) for 1 h at room temperature. Immunoreactive protein bands were developed using HRP Substrate Luminol Reagent (Millipore Corporation, Billerica, MA 01821 USA) and band intensities analyzed using a bio-Imaging system.
β actin
Manufactured by Abbkine
Sourced in United States, China
β-actin is a widely used internal control protein for Western blotting and other protein analysis techniques. It is a critical structural component of the cytoskeleton and is involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Mtor p mtor, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Hrp substrate luminol reagent, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
Amersham imager 600 system, by GE Healthcare (1 mentions)
Ab115730, by Abcam (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Peroxidase conjugated secondary antibody, by Abbkine (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Txnip, by Proteintech (1 mentions)
Ecl western blotting substrate, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Keap1, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Western blot apparatus, by Bio-Rad (1 mentions)
14 protocols using β actin
1
Protein Expression Analysis in HUVECs
2
Protein Expression Analysis via Western Blot
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Tissues and cells were lysed with a modified buffer, and western blotting was performed as described previously41 (link). The primary antibodies were as follows: MLXIPL (Abcam, ab92809), GLUT1 (Abcam, ab115730), PKM1 (Abcam, ab116271), PKM2 (Abcam, ab137852), LDHA (Abcam, ab84716), and β-actin (Abbkine, A01011). And images were captured using an Amersham Imager 600 System (GE Healthcare).
3
Western Blot Analysis of 5-HT1A in HEK-293 Cells
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Total protein from HEK-293, HEK-293+pEGFP-N1-Basic and HEK-293+pEGFP-N1-CEBPB cells was extracted by NP-40 and PMSF. Protein from SK-N-SH cells was collected as described. Quantified protein samples were separated by electrophoresis in 10% denatured polypropylene gel and transferred using polyvinylidene fluoride (PVDF) membrane for 1 h at 100 V. After blocking with 8% skimmed milk, primary antibodies for 5-HT1A (Thermo Scientific™) and βactin (Abbkine, CA, USA) were diluted with TBS-T at a ratio of 1:1000 and 1:2000, respectively. Membranes were incubated overnight at 4 °C. Diluted secondary antibody (Abbkine) (1:5000) was incubated with the membrane, which had been washed three times with TBS-T for 2 h. Target protein expression was detected by Lumino (ECL)luminescent solution and the Tanon-5500 chemiluminescence imaging analysis system. Each sample was tested in duplicate per experiment, and three separate experiments were conducted.
4
Western Blot Analysis of VWF and SPP1
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Cells were lysed in IP lysis buffer containing protease inhibitors and then measured protein concentration by BCA assay. An equal amount of protein from cells loaded into the 8% SDS-PAGE, transferred to polyvinylidene difluoride membranes (PVDF), then incubated with primary antibody against VWF (1:500, Cell Signaling Technology, Shanghai, China) and SPP1 (1:2,000, Proteintech, Wuhan, China) at 4°C overnight, followed by incubation with peroxidase-conjugated secondary antibody (1:5,000, Abbkine, Wuhan, China) at room temperature for 1 h, finally visualized by enhanced chemiluminescence detection. The β-actin (1:5,000, Abbkine, Wuhan, China) was used as internal references in this study.
5
Western Blot Analysis of Skin Wound Proteins
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Protein samples were extracted from skin wound tissues or cultured cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane. Membranes were probed with primary antibodies against TXNIP (Proteintech, Chicago, Illinois, United States), NLRP3, caspase-1, IL-1β, phosphorylated Akt ser473 (p-Akt), Akt, phosphorylated GSK3β ser9 (p-GSK3β), GSK3β (Cell Signaling Technology, Beverly, Massachusetts, United States) and β-actin (Abbkine, Redlands, California, United States). Membranes were then washed and labeled with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Abbkine). β-actin and all secondary antibodies were used at a dilution of 1:10,000. All other antibodies were used at a dilution of 1:1,000.
6
Western Blot Analysis of Autophagy Markers
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After being treated according to their groups, the cells were harvested and lysed. The supernatant was mixed with sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min. Equal amounts of protein were separated by SDS−polyacrylamide gel electrophoresis (SDS–PAGE), followed by semidry blotting on a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). After blocking the membrane with 5% (w/v) Tris-buffered saline with Tween 20 (TBST) and non-fat dry milk, the primary antibodies were added. The secondary antibodies—HRP-conjugated anti-mouse/anti-rabbit IgG (GenScript, Nanjing, China)—were detected by enhanced chemiluminescence (ECL) using an ECL Western Blotting Substrate (Bio-Rad, Hercules, CA, USA). The antibodies used were as follows: LC3B (CST, Beverly, MA, USA); p62 (Proteintech, Wuhan, China); KEAP1 (Proteintech, Wuhan, China); NRF2 (Proteintech, Wuhan, China); and β-actin (Abbkine, Wuhan, China). The band densities were analyzed using the software ImageJ.
7
Western Blot Analysis of TGF-β Signaling
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Whole-cell lysates were prepared from siFAT1/siControl-transfected cells at 72 h using RIPA buffer (Thermo Fisher Scientific), protease inhibitor cocktail (Sigma-Aldrich), and phosphatase inhibitor cocktail (Sigma-Aldrich). In this study, 40–100 µg of lysates were loaded in 5%–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and electrophoresed in Western blot apparatus (Bio-Rad). Primary antibodies used were as follows: TGF-β1 (rabbit polyclonal, 1:1,000, Abcam, #ab92486), TGF-β2 (mouse monoclonal, 1:1,000, Abcam, #ab36495), Serpine1/PAI-1 (rabbit polyclonal, 1:1,000, Abcam, #ab66705), SMAD2/3 (rabbit monoclonal, 1:1,000, Cell Signaling Technology, #8685), and β-actin (mouse monoclonal, 1:5,000, Abbkine).
8
Western Blot Analysis of CD206 and TREM2
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The BV-2 cells were homogenized with RIPA buffer and protease inhibitors (Thermo Scientific). Equal amounts of protein (40 μg/lane) were loaded to gradient (4 to 15%) concentrations of polyacrylamide SDS-PAGE (Smobio, Hsinchu City, Taiwan) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) using an electro-transfer system (Bio-Rad, Hercules, CA). The membranes were blocked with 5% nonfat milk and probed with the following primary antibodies for 16 h at 4 °C: rabbit polyclonal anti-CD206 (1:1,000; Abbkine, Wuhan, China), goat polyclonal TREM2 (1:1,000, ThermoFisher Scientific), and mouse monoclonal β-actin (1:5,000; Abbkine). The membranes were incubated with the appropriate secondary antibodies (Bethyl, Inc., Montgomery, TX, USA) conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Bound antibodies were visualized with D-Plus ECL Pico System (Dongin LS, Republic of Korea) and a G: Box Chemiluminescence & Fluorescence system (Syngene, Frederick, MD, USA).
9
Spinal Cord Protein Expression Analysis
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Total protein extract was prepared from T8-12 spinal cord tissues with protein lysis buffer. Western blotting was performed as described previously [65 (link), 66 (link)]. 50μg of protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Then, the PVDF membrane was blocked with 5% skimmed milk in TBST for 1h at room temperature. Then, the membranes were incubated with the following primary antibodies: P2×7R (Boster, China), S100A9 (Boster, China), Akt (Cell Signaling Technology, USA), P-Akt (Cell Signaling Technology, USA), Bcl-2 (Affinity Biosciences, USA), Bax (Affinity Biosciences, USA), and Caspase-3 (ABclonal, USA). Further, after washes with 1X TBST, the membrane was incubated with anti-mouse or goat HRP-conjugated secondary antibodies (Abbkine, USA). This was followed by protein detection by ECL Assay Kit (Bipec Biopharma). β-actin (Abbkine, USA) was used as internal control. Band intensity was quantified using ImageJ software (1.44 P) and the expression of target proteins relative to β-actin control was determined.
10
Western Blot Analysis of Protein Expression
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Cells were homogenized with RIPA buffer (50 mM Tris‐HCl (pH 7.4), 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Na‐deoxycholate and 1% NP‐40) supplemented with cocktail protease inhibitor (1:500; Roche, Castle Hill, NSW, Australia). All homogenates were centrifuged at 15,000 RCF for 30 min at 4°C to collect the supernatants and total protein concentration was assessed by BCA assay (Beyotime Biotechnology). After boiling at 100°C for 5 min, 12% SDS‐polyacrylamide gels were used to separate 30 µg protein per well followed by a semi‐dry transfer for 20 min. After blocking with 5% nonfat milk, membranes were probed with primary antibodies (IL‐1β, USCN, cat# PAA563Rb51, rabbit, 1:2,000; AKT, Bioss, cat#bs‐0115R, rabbit, 1:500; phospho‐AKT, Bioss, cat#bs‐5194R, rabbit, 1:500) overnight at 4°C. β‐actin (Abbkine, cat# A01010, mouse, 1:2,000) was used as a loading control. After incubation with appropriate secondary antibodies (HRP, Goat Anti‐Mouse IgG, cat# A21020, 1:5,000; HRP, Goat Anti‐Mouse IgG, cat# A21010, 1:5,000) for 1 hr at room temperature, protein bands were visualized and analyzed using a chemiluminescent imaging system (Bio‐Rad).
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