Cells were lysed in ice-cold RIPA cell buffer (P0013B, Beyotime Biotechnology, China) supplemented with protease inhibitor cocktail. The proteins (40–100 μg) were separated on 10–15% PAGE gels and electrotransferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk, the membrane was incubated with specific primary antibody and horseradish peroxidase-conjugated secondary antibody. The antibodies used are Flag (GNI4110-FG-S, GNI, Japan, 1:1000), HA (GNI4110-HA-S, GNI, Japan, 1:1000), Stat3 (12640S, Cell Signaling Technology, USA,1:1000), phospho-Stat3Y705 (9131S, Cell Signaling Technology, USA, 1:1000), Socs3 (sc-73045, Santa Cruz, 1:1000), Prdm14(D221722, BBI, China, 1:1000), H3K27me3 (39055, Active Motif, China, 1:1000), H3 (4620s,Cell Signaling Technology, USA, 1:1000) and β-Tubulin (200608, ZENBIO, China, 1:2000). The band density was analyzed with ImageJ according to ImageJ User Guide. Briefly, we inverted the greyscale images and select sample bands with rectangular selections. The proteins levels were normalized with respect to the β-Tubulin level, and the grayscale ratio of protein/β-Tubulin was calculated and visualized with GraphPad Prism 8.0.
Ripa cell buffer
Manufactured by Beyotime
Sourced in China
RIPA cell buffer is a lysis buffer used to extract proteins from cells. It contains a combination of detergents and salts that disrupt cell membranes and solubilize cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Phospho stat3 y705, by Cell Signaling Technology (1 mentions)
H3k27me3, by Active Motif (1 mentions)
β tubulin, by ZenBio (1 mentions)
β tubulin, by GraphPad (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Socs3, by Santa Cruz Biotechnology (1 mentions)
Prism 8, by GraphPad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Ab24741, by Abcam (1 mentions)
Sc 8035, by Santa Cruz Biotechnology (1 mentions)
4 protocols using ripa cell buffer
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Signaling Pathways
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CD19+ B cells were challenged by LPS for 6 h with/without Wogonin (12.5, 25, and 50 μM). Cells were lysed in RIPA cell buffer (Beyotime) supplemented with PMSF (Solarbio) and protease inhibitors (Roche) on ice for 30 min. 50 μg of protein sample in the lysate was separated in 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked in TBST (Triton-containing Tris-buffered saline) with 5% milk for 2 h at room temperature and then incubated with specific primary antibodies overnight at 4°C. The secondary antibodies were added to the membranes and incubated for 1 h. Blot bands were visualized by enhanced chemiluminescence. The quantitative analysis was evaluated using ImageJ software according to the grey value of western blot bands.
The primary antibodies were used including Phospho-STAT3 (p-STAT3), total STAT3 (t-STAT3), Phospho-AKT (p-AKT), total AKT (t-AKT), Phospho-ERK (p-ERK), total ERK (t-ERK), Hif-1α (all were from Cell Signaling Technology), and GAPDH (Santa Cruz Biotechnology). The secondary antibodies were also purchased from Cell Signaling Technology.
The primary antibodies were used including Phospho-STAT3 (p-STAT3), total STAT3 (t-STAT3), Phospho-AKT (p-AKT), total AKT (t-AKT), Phospho-ERK (p-ERK), total ERK (t-ERK), Hif-1α (all were from Cell Signaling Technology), and GAPDH (Santa Cruz Biotechnology). The secondary antibodies were also purchased from Cell Signaling Technology.
3
Protein Expression Analysis by Western Blot
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Cells were lysed in pre-cooled RIPA cell buffer (P0013B, Beyotime Biotechnology, China) containing protease inhibitor (DI111-02, TRANSGEN BIOTECH, China, 1: 100). Proteins were separated by a 10% SDS-PAGE gel at RT and electrotransferred to a PVDF membrane in ice-bath. Probing was performed with specific primary antibodies and HRP-conjugated secondary antibodies. The primary antibodies used were PIAS1 (ab2474-1, Abcam, 1:1000) and GAPDH (SC-8035, Santa Cruz, 1:2000). Proteins were detected in super ECL detection reagent (No.180-501, Tanon, China).
4
Western Blot Analysis of EMT Markers
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Cells were lysed in ice-cold RIPA cell buffer (P0013B, Beyotime Biotechnology, China) supplemented with protease inhibitors cocktail. The protein samples were then separated on a 10%- or 15%-PAGE gel and electrotransferred to a PVDF membrane. After blocking, the membrane was incubated with specific primary antibodies overnight at 4 °C and followed by a HRP-conjugated secondary antibody at room temperature. Images were captured under the Chemiluminescence Gel Imaging System Tanon-5200Multi (Shanghai Tianneng, China). The primary antibodies are FLAG (SG110-26, GNI, Japan, 1:1000), Nanog (14295-1-AP, Proteintech, USA, 1:1000), Gadd45g (SC-33173, Santa Cruz, USA, 1:500), Gadd45a (UPA06635, Gene Universal, China, 1:500), Gadd45b (UPA01987, Gene Universal, China, 1:500), MEK1/2 (380797, ZENBIO, China, 1:1000), Phospho-MEK1/2 (Ser217/221) (310050, ZENBIO, China, 1:1000), ERK1/2 (201245-4A4, ZENBIO, China, 1:500), Phospho-ERK1 (Thr202/Tyr204)/ERK2 (Thr185/Tyr187) (301245, ZENBIO, China, 1:500), Raf1 (251817, ZENBIO, China, 1:1000), Phospho- Raf1 (Ser338) (D155090, BBI, China, 1:1000), β-tubulin (200608, ZENBIO, China, 1:2000), E-cadherin (201283, ZENBIO, China, 1:1000), and N-cadherin (383341, ZENBIO, China, 1:1000).
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