Cells in each group were washed with ice-cold phosphate-buffered saline (PBS) three times and lysed in RIPA solution containing protease inhibitor phenylmethanesulfonyl fluoride (PMSF), phosphatase inhibitor NaF and Na3VO3. Protein concentration was measured by BCA method. 50 μg total protein was added in 10% sodium dodecyl sulfate-polyacrylamide gel and then transfered to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 1 hour and incubated with specific antibodies(polyclonal antibody against p-p38α MAPK; 1:1000; Millipore, USA; polyclonal antidoby against p38α MAPK, HA-Tag; 1:1000; CST, USA; monoclonal antibody against Myc-Tag; 1:1000; CST, USA; polyclonal antibody against RARα; 1:1000; Santa Cruz, USA; polyclonal antibody against C/EBPβ, CD11b; 1:500; Wanleibio; China) overnight at 4 °C and then with secondary antibody(goat anti-rabbit antibody, 1:5000 and goat anti-mouse antibody, 1:2000; Zhongshan Goldenbridge Biotechnology Co. Ltd., Beijing, China) for 1 h at 37 °C. After washing with Tris-Buffered Saline Tween-20 and Tris-Buffered Saline (TBST and TBS), the autoradiograms were scanned and subjected to densitometry. β-actin (monoclonal antibody against β-actin, 1:1000; Zhongshan Goldenbridge Biotechnology Co. Ltd.,Beijing, China) was used as an internal control.
β actin
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China, United States
β-actin is a cytoskeletal protein found in all eukaryotic cells. It is a component of the microfilament system and plays a crucial role in maintaining cell structure and facilitating cellular processes such as cell motility, division, and signaling. β-actin is commonly used as a reference gene or loading control in various molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (8 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
U0126, by Merck Group (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Apaf 1, by Santa Cruz Biotechnology (2 mentions)
Sc 271759, by Santa Cruz Biotechnology (2 mentions)
Caspase 3, by Santa Cruz Biotechnology (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Cytochrome c, by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Zb 2301, by Zhongshan Golden Bridge Biotechnology (2 mentions)
Zb 2305, by Zhongshan Golden Bridge Biotechnology (2 mentions)
Electrochemiluminescence reagent, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (2 mentions)
35 protocols using β actin
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of Cell Signaling
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After washing with cold PBS, cells were lysed in RIPA supplemented with PMSF (Beyotime Biotechnology, Shanghai, China) protease inhibitor and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). Lysates were adjusted to similar concentrations, mixed with 6× sample loading buffer and boiled for 5 min. The samples resolved by 4–20% SDS-PAGE gradient gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp, MA, USA). After blocked with 5% skimmed milk, the membranes were incubated with primary antibodies (YAP, p-YAP, Glut1, HKII, LDHA ERK1/2, p-ERK1/2, JNK, p-JNK, p38 and p-p38; Abcam, Cambridge, USA) (β-Actin; Zhongshan Golden Bridge Biotechnology, Beijing, China) overnight at 4 °C. Membranes were incubated with appropriate secondary antibodies (Zhongshan Golden Bridge Biotechnology, Beijing, China) for 1 h at room temperature, followed by detection using an ECL blotting analysis system (Bio-Rad, CA, USA).
3
Western Blot Analysis of Liver Proteins
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Total protein was extracted from mouse livers or HepG2 cells using Roth lysis buffer containing proteinase inhibitor cocktail (Applygen Technologies Inc, China) and centrifuged for 10 minutes at 4°C at 12 000 g to collected supernatants. 20‐80 μg total protein was separated by 10%‐12% SDS‐PAGE. Primary antibodies were incubated in 5% milk at 4°C overnight. Proteins examined in this study include PANDER (ABclonal, A13592), FOXO1 (Cell Signaling technology, 2880S), phosphorylated FOXO1 (Ser256, Cell Signaling technology, 9461S), PEPCK (Bioworld, BS6870), G6Pase (Santa Cruz, sc‐25840), COX 4 (Huaxingbio, HX1842), Lamin B1 (Huaxingbio, HX‐1846), β‐actin (Zhongshan Golden Bridge, TA‐09). ImageJ (version 1.42) was used to analyse protein expression, and data were normalised to β‐actin protein expression.
4
Spinal Cord Injury Signaling Pathways
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Protein lysates were extracted from tissues of injured spinal cord tissue 72 hrs post-injury. Protein concentration was determined using the BCA protein assay kit (CW0014S, CWBIO; Haimen, Jiangsu, China). Concentrations of 20–40 µg total protein were separated on polyacrylamide gels, transferred to polyvinylidene difluoride membranes and incubated overnight at 4°C with the following primary antibodies: NF-κB (p65) (10745-1-AP, Proteintech), p-NF-κB (Ser536) (#3033, Cell Signaling), IκBα (10268-1-AP，Proteintech Group), p-IκBα (#2859, Cell Signaling), and β-actin (TA-09, Zhongshan Golden Bridge Biotechnology, Beijing, China). Membranes were incubated with secondary antibodies, and signals were visualized using enhanced chemiluminescence.
5
Shikonin-induced Apoptosis Signaling
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Shikonin and dimethylsulfoxide were purchased from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The purity of Shikonin was >98%. Antibodies against caspase-3, poly ADP-ribose polymerase (PARP), c-Myc, p-ERK1/2, ERK1/2, p38 MAPK, p-JNK and JNK were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against p-p38 MAPK was purchased from Merck KGaA. Goat anti-rabbit, goat anti-mouse and β-actin antibodies were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).
6
Protein Expression Analysis in Liver Cancer
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As usual, proteins from liver tissues or HCC cells were lysed for 30 min on ice, and centrifuged at 14,000×g for 20 min at 4°C. The supernatant was collected to detect the expression of proteins. The proteins were resolved by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). Membranes were blocked with 5% dried skimmed milk in 0.05% Tween 20-PBS for 2 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: GRK2, GRK3, EP2 (Santa Cruz Biotechnology, CA, USA), β-actin (Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China), Na+/K+ ATPase (Abcam, Cambridge, UK), phospho-Akt (p-Akt), Akt, phospho-ERK (p-ERK) and ERK (Cell Signaling, MA, USA). The membranes were treated with the secondary antibodies followed by further incubation for 2 h. Proteins were quantitated with Image Quant LAS 4000mini (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed by ImageJ software version 1.4.2b (NIH, Bethesda, MD, USA). β-actin and Na+/K+ ATPase expressions were used as a loading control for total and membrane fractions, respectively.
7
Elucidating Gem-Induced Apoptosis Mechanisms
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To reveal the mechanism of the apoptotic effect of gem, Western blotting was done for apoptotic-related proteins such as c-IAP2 and Bcl-2. Briefly, after treatment with indicated doses of gem for 48 h, cells were collected and lysed with TRIzol for extracting total protein. The protein lysates were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were soaked in blocking buffer (50 g/L bovine serum albumin dissolved in tris-buffered saline) for 1 h. To probe for c-IAP2 and Bcl-2, membranes were incubated overnight at 4°C with relevant antibodies (1:200 dilution), followed by appropriate horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection. β-actin (1:400 dilution) purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China) was used as an internal control. Semi-quantification analysis of Western blottings was done by Image-pro plus 6.0 software (http://www.mediacy.com).
8
Molecular Mechanisms of HMGB1 Signaling
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The following reagents were used: rHMGB1 (Cat No. #1690-HMB-050, R&D Systems, Minneapolis, MN, USA), G1 (Cat. No. 3577/10, TOCRIS, Bioscience, Bristol, UK), G15 (Cat. No. 3678/10, TOCRIS, Bioscience, Bristol, UK). E2, TAM, were obtained from Sigma–Aldrich (St. Louis, MO, USA). U0126, and the LY294002 were purchased from Millipore (Temecula, CA, USA). E2 was dissolved in ethanol and other drugs were solubilized in dimethyl sulfoxide (DMSO; Sigma–Aldrich). The following antibodies were used: anti-HMGB1 neutralizing antibody (Cat No. H00003146-M08, R&D systems, Minneapolis, MN, USA); p-ERK1/2 (Cat No. AP0484P, diluted 1:1000), ERK1/2 (Cat No. BS90472, diluted 1:1000), Akt (Cat No. BS6473, diluted 1:1000) were purchased from Bioworld (St Louis Park, MN, USA). p-Akt (Cat No. 4060, Cell signaling Technology, USA, diluted 1:1000), LC3B (Cat No. 2775 Cell Signaling Technology, diluted 1:1000), P62 (Cat No. 5114, Cell Signaling Technology, diluted 1:1000); Beclin1(Cat No. 210498, diluted 1:1000), GPR30 (Cat No. ab39142, diluted 1:250), HMGB1 (Cat No. ab18256, diluted 1:500), Bcl-2 (Cat No.32124, diluted 1:1000), BAX (Cat No. 182734, diluted 1:1000) were purchased from Abcam (Cambridge, MA, USA), and β-actin (Cat No. TA-09, diluted 1:1000) from Zhongshan Golden Bridge (Beijing, China).
9
Apoptotic Protein Expression Analysis
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The expression of apoptotic proteins of each group was analyzed by Western blot as previous described using specific antibody against Caspase-3 (sc-271759, 1:100, Santa Cruz Biotechnology Inc.), cleaved Caspase-3 (asp175) (no 9661, 1:1,000, Cellsignal), Bcl-2 (sc-7382, 1:250, Santa Cruz Biotechnology Inc.), Apaf-1 (sc-135625, 1:500, Santa Cruz Biotechnology Inc.), Cytochrome C (sc-13156, 1:200, Santa Cruz Biotechnology Inc.), Bax (sc-70406, 1:200, Santa Cruz Biotechnology Inc.) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology). The experiments were repeated at least three times independently.
10
Rat Heart Tissue Protein Profiling
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Rat heart tissue (100 mg) was lysed with 1 ml of Radio‐Immunoprecipitation Assay (RIPA) and 10 μl of Phenylmethanesulfonyl Fluoride (PMSF) in an eppendorf tube using an ultrasonic tissue disrupter. Protein concentrations were measured using the Bicinchoninic Acid (BCA) assay. Protein samples (40 μg) were separated by SDS‐PAGE gel electrophoresis and transferred onto a nitrocellulose membrane. Membranes were then blocked in 5% skim milk for 1 hr, followed by incubation with primary antibody against Cyt‐c (1:1000; Cell Signaling Technology, Inc., Shanghai, China), caspase‐3 (Cell Signaling Technology, Inc.), Bax, Bcl‐2 and p‐JNK overnight at 4°C. Membranes were washed three times with Tris Buffered Saline with Tween20 (TBST), followed by incubation with secondary antibody (1:5000; Zhongshan Golden Bridge, goat anti‐rabbit lgG‐HRP) for 1 hr. β‐actin (1:1000; Zhongshan Golden Bridge, Beijing, China) was used as the internal control. Specific bands were detected using an enhanced chemiluminescence system and captured on X‐ray film. Western blots were performed in at least three independent experiments. The density of the bands on the membrane was scanned and analysed with Quantity one software (Life Science Research, Education, Process Separations, Food Science, Hercules, California).
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