ADSCs were pretreated with PD98059 (HY-12028, 80 μM, MedChemExpress), GsMTx4 (HY-P1410A, 5 μM, MedChemExpress), and Yoda1 (HY-18723, 30 μM, MedChemExpress) for 1 hour as requested, and the cells were harvested 2 hours after LIPUS stimulation without specification. ADSCs were lysed on ice with RIPA buffer (Beyotime, Shanghai, China) containing the protease inhibitor PMSF (Beyotime, Shanghai, China) and Phosphatase Inhibitor Cocktail (YEASEN, Shanghai, China). The protein concentrations of cell lysates were measured using a BCA protein assay kit (Beyotime, Shanghai, China). Protein samples (15 μg) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 10% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were ERK (ab184699, 1 : 10000, Abcam, USA), p-ERK (ab76299, 1 : 5000, Abcam, USA), VEGFA (ab214424, 1 : 1000, Abcam, USA), and GAPDH (ab181602, 1 : 10000, Abcam, USA). After being rinsed with TBST, the membranes were hybridized with secondary antibodies and reacted with ECL solution (NCM Biotech Co., Ltd, Suzhou, China). The chemiluminescent images were taken, and the density of each protein band was determined using ImageJ software.
Ab76299
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab76299 is a laboratory equipment product offered by Abcam. It serves as a core component for conducting various scientific experiments and research activities. The detailed specifications and technical details of this product are not available for an unbiased and factual description within the given constraints.
Automatically generated - may contain errors
Lab products found in correlation
Ab184699, by Abcam (11 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab170099, by Abcam (2 mentions)
Ab81283, by Abcam (2 mentions)
Ab178876, by Abcam (2 mentions)
Ab124956, by Abcam (2 mentions)
Ma1 110, by Thermo Fisher Scientific (2 mentions)
Ecl system, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab214424, by Abcam (1 mentions)
Phosphatase inhibitor cocktail, by Yeasen (1 mentions)
Ab181602, by Abcam (1 mentions)
Gsmtx4, by MedChemExpress (1 mentions)
Yoda1, by MedChemExpress (1 mentions)
Pd98059, by MedChemExpress (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ab205718, by Abcam (1 mentions)
16 protocols using ab76299
1
LIPUS Stimulation of ADSC Signaling
2
Western Blot Analysis of RAF, ERK1/2, and p-ERK1/2
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Proteins were extracted from MH7A cells, ankle joint and knee joint tissues using RIPA lysis buffer (BeyoTime Biotech, Shanghai, China) and quantified by a BCA Protein Assay Kit (BeyoTime, China). A total of 20 μg protein sample was separated by 8% SDS-PAGE and blotted onto PVDF membrane. After blocked with 5% non-fat milk in TBST for 1 h at room temperature, the membranes were then incubated with appropriate primary antibodies at 4 °C overnight, followed by probed with secondary antibody (Abcam, USA, ab205718) conjugated to horseradish peroxidase. The protein signals were visualized using an ECL detection system. The primary antibodies including RAF (Abcam, USA, ab170099), ERK1/2 (Abcam, USA; ab184699), p-ERK1/2 (Abcam, USA, ab76299).
3
Protein Expression Analysis in Leukemic Cell Lines
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Cells were lysed with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Biological Reagent Co., Ltd., Shanghai, P.R. China) supplemented with protease inhibitor phenylmethane sulfonyl fluoride (PMSF; Cell Signaling Biological Reagent Co., Ltd.). A BCA protein assay kit (Beyotime Biotechnology, Shanghai, P.R. China) was used to measure the concentration of protein samples. Protein (50 μg) from NB4 and HL-60 cells was loaded onto every lane. The following primary antibodies were used for immunoblotting: β-actin (1:1,000; Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd, Beijing, P.R. China), RREB1 (AP19046b; 1:1,000; Abgent, San Diego, CA, USA), CEBPβ (WL0056a; 1:1,000; Wanleibio, Co., Ltd., Beijing, P.R. China), CD11b (ab133357; 1:1,000; Abcam, Cambridge, UK), ERK (ab184699; 1:1,000; Abcam), p-ERK (ab76299; 1:1,000; Abcam), AKT (ab8805; 1:1,000; Abcam), pAKT (ab81283; 1:1,000; Abcam). The chemiluminescence reaction was performed using Pierce ECL West Femto substrate (Thermo Fisher Scientific Co., Ltd, Shanghai, P.R. China), and images were captured using an Image Lab system (Bio-Rad Laboratories, Inc.).
4
Antibody-based Signaling Pathway Analysis
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Antibodies against PCNA (2586), phosphor-MEK1/2 (9154), phosphor-p90RSK (11989) and phosphor-MSK1 (9595) were obtained from Cell Signaling Technology (CST, MA, USA). Antibodies against AdipoR1 (ab126611), p90RSK (ab32114), cyclinD1 (ab134175), CDK4 (ab108357), CDK6 (ab124821), total-ERK1/2 (ab184699), phospho-ERK1/ (ab76299) and total-MEK1/2 (ab178876) were obtained from Abcam (Cambridge, UK). Recombinant human adiponectin (1065-AP) and recombinant human epidermal growth factor (EGF, 236-EG) were obtained from R&D System.
5
Protein Expression in ccRCC Cells
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Total protein of ccRCC cells and tissues is obtained using RIPA lysis buffer containing protease inhibitors, and BCA protein assay Kit is used to quantitate the protein levels. Then, Western Blot is used to detect the protein expression of ZNF582, TJP2, ERK2, p-ERK2, MEK1/2, p-MEK1/2, BCL-2, Caspase-3, Cleaved Caspase-3, E-cadherin and N-cadherin in these samples. The details of these antibodies were as follows: anti-ZNF582 (1:1000, SAB1408372, Sigma), anti-Flag (1:1000, 14793 S, CST), anti-TJP2 (1:1000, 18900-1-AP, Proteintech), anti-ERK2 (1:1000, ab32081, Abcam), anti-ERK1 (pT202/pY204) + ERK2 (pT185/pY187) (1:5000, ab76299, Abcam), anti-MEK1 + MEK2 (1:20000, ab178876, Abcam), anti-MEK1 + MEK2 (phospho S217 + S221) (1:5000, ab278723, Abcam), anti-BCL-2 (1:2000, 12789-1-AP, Proteintech), anti-Caspase-3 (1:1000, 9662 S, CST), anti-Cleaved Caspase-3 (1:1000, AF7022, Affinity), anti-E-cadherin (1:10000, ab40772, Abcam), anti-N-cadherin (1:1000, 9664 S, CST) and anti-GAPDH (1:8000, 10494-1-AP, Proteintech).
6
Comprehensive Protein Analysis by Western Blot
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Proteins were extracted with radioimmunoprecipitation (RIPA) lysis buffer with PMSF, quantified by BCA Protein Assay Kit (Beyotime). Western blot was performed in triplicates according to the procedures reported previously [24 (link)]. GAPDH was used as an internal control. We used the following antibodies: p-AKT (1:2000; rabbit IgG, CST, 4060T), p-ERK1/2 (1:5000; rabbit IgG, Abcam, ab76299), p-STAT5α (1:1000; rabbit IgG, Abcam, ab30648), p-GSK (1:5000; rabbit IgG, Abcam, ab75814), AKT (1:1000; mouse IgG, proteintech, 60203-2-Ig), ERK1/2 (1:1000; rabbit IgG, proteintech, 16443-1-AP), STAT5α (1:1000; rabbit IgG, Abcam, ab32043), GSK3β (1:1000, rabbit IgG, proteintech, 22104-1-AP), CD63 (1:500; rabbit IgG, proteintech, 25682-1-AP), TSG101 (1:500; rabbit IgG, Abcam, ab83), HSP70 (1:100; rabbit IgG, SBI, EXOAB-KIT-1), calnexin (1:2000; rabbit IgG, CST, 2433s), GAPDH (1:10000; rabbit IgG, proteintech, 10494-1-AP) (1:10000; mouse IgG, proteintech, 60004-1-Ig), HRP-conjugated anti-rabbit-IgG (NeoBioscience), HRP-conjugated anti-goat-IgG (NeoBioscience), and HRP-conjugated anti-mouse-IgG (NeoBioscience).
7
Detailed Synthesis and Characterization of DHP1808
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The preparation of DHP1808 was as per our previous reports [38 (link)]. VX-765 and Z-VAD-FMK were obtained from Selleckchem Co. Ltd. (Shanghai, China). The antibodies recognizing FADD (14906-1-AP), Bcl-2 (12789-1-AP), Bax (50599-1-AP), Cytochrome C (10993-1-AP), β-Catenin (51067-2-AP), CDK2 (10122-1-AP), CDK4 (11026-1-AP), CDK6 (14052-1-AP), CyclinB1 (55004-1-AP), p21 (10355-1-AP), E-Cadherin (20874-1-AP), MMP2 (10373-1-AP), MMP9 (10375-1-AP), ZEB1 (21544-1-AP), GAPDH (60004-1-Ig) and β-actin (60008-1-Ig), MNK1 (10136-1-AP), MDM2 (19058-1-AP), and p53 (10442-1-AP) were purchased from Proteintech (Wuhan, China). The antibody recognizing Fas (Ab82419), FasL (Ab68338), Caspase-3 (Ab13847), PARP (Ab32138), N-Cadherin (Ab76011), Met (Ab51067), Phos-Met (Ab68141), ERK1/2 (Ab17942), Phos-ERK1/2 (Ab76299), JNK (Ab179461), Phos-JNK (Ab124956), phos-MNK1 (ab109102), Caspase-1 (ab179515), and GSDMD (ab215203) were purchased from Abcam (Cambridge, MA, USA). The antibody recognizing Bad (9239), Bim (2933), Caspase-8 (9746), Caspase-9 (9508), p27 (3686), Cdc37 (10218-1-AP), cRaf (2330), Phos-cRaf (2330), BRaf (2330), Phos-bRaf (2330), c-Myc (5605), p90RSK (9326), Phos-p90RSK (9326), Hsp90 (4877), EGFR (2232), Phos-EGFR (4407), Akt1 (4691), phos-Akt308 (4056), and phos-Akt473 (4060) were purchased from Cell Signaling Technology (Danvers, MA, USA).
8
Oxidative Stress and Apoptosis Regulation
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Apocynin was supplied by Selleck Chemicals (Houston, TX, USA). Sodium taurocholate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific for NADPH oxidase 2 (Nox2/gp91phox, ab31092), NADPH oxidase 4 (Nox4, ab133303), Bax (ab32503), p38 (ab170099), p-p38 (Thr180/Tyr182, ab4822), ERK1/2 (ab184699), p-ERK1/2 (Thr202/Tyr204 and Thr185/Tyr187, ab76299), and p-JNK (Thr183/Thr221, ab124956) were purchased from Abcam (Cambridge, MA, USA). Antibody for caspase-3 (# 9662S) was purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for Bcl-2 (GTX100064) was purchased from GeneTex Inc. (Irvine, CA, USA). Antibodies for JNK and GAPDH were purchased from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijing, China). NADPH-Na4 was supplied by Beijing Solarbio Science and Technology Co. Ltd. (Beijing, China). Dihydroethidium (DHE) was purchased from Beyotime Biotechnology (Shanghai, China). All other chemicals used in this study were of analytical grade and were commercially available.
9
Western Blot Analysis of Neuronal Signaling
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Cellular lysates from cultured neurons were prepared using RIPA buffer (ThermoFisher Scientific) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (ThermoFisher Scientific). After measuring protein concentration with the BCA protein assay kit (Pierce, Rockford, IL), protein samples were loaded to 10% SDS-polyacrylamide gels. After separation, proteins were transferred onto polyvinylidene difloride (PVDF) membranes. The PVDF membranes were blocked with 5% bovine serum albumin and then probed with primary antibodies (phospho-ERK1/2, Abcam, ab76299, 1:5000 dilution; ERK1/2, Abcam, ab184699, 1:5000 dilution; phospho-CREB, Abcam, ab32096, 1:5000 dilution; CREB, Cell Signaling Technology, 9197, 1:1000 dilution) at 4 °C overnight. Membranes were then incubated with peroxidase-conjugated secondary antibody for 1 h; detection was performed using West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific) and scanned with an Amersham Image 600 imager. Densitometry analysis was done with ImageJ.
10
Brain Protein Extraction and Western Blot
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Total protein from brain tissues was extracted by using RIPA Lysis Buffer (P0013, Beyotime, Shanghai, China) with 1 mM PMSF following the manufacturer’s instructions. 30 g proteins were boiled at 100°C for each sample with protein loading buffer for 5 min, followed by separation in 10–12% SDS-PAGE electrophoresis and transferred onto PVDF membranes. Then, 5% lipid-free milk/TBST buffer were used to block the membranes at room temperature overnight, incubated with anti-p-ERK1 + anti-ERK2 (ab76299, 1:5000, Abcam), anti-CREB (ab32515, 1:1000, Abcam), anti-BNDF (ab108319, 1:1000, Abcam) and anti-GAPDH (ab8245, 1:5000, Abcam) primary antibodies for 2 h at 4°C overnight, respectively. After being incubation with secondary antibodies anti-mouse IgG (BA1051, 1:20,000, BOSTR) or anti-rabbit IgG (BA1054, 1:20,000, BOSTR,) for 1–2 h at room temperature, the immuno-complexes were finally detected by ECL after washing by TBST and analyzed using the Image-Pro Plus 6.0 software [30 (link)].
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