Proteins were extracted using radioimmunoprecipitation buffer (Kangwei Century Biotechnology Co., Ltd.), and protein concentrations were measured using a bicinchoninic acid (BCA) assay (Kangwei Century Biotechnology Co., Ltd., Beijing, China). Equal amounts of clear lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes (Immobilon P, Millipore, Bedford, MA, USA). Equal transfer was validated by staining with Ponceau red. The membranes were blocked with 10% skim milk in Tris-buffered saline (TBS) and then incubated with primary antibodies in TBS containing 0.05% Tween 20, 2% bovine serum albumin, and 0.05% sodium azide overnight at 4 °C. The following antibodies were used at the indicated dilutions: RIP140 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 1:1,000 and β-actin at 1:10,000 (ProteinTech Group, Inc., Wuhan, China). Secondary horseradish peroxidase-conjugated antibodies (rabbit, ProteinTech Group, Inc., Wuhan, China) were used at 1:10,000 in 10% skim milk in TBS containing 0.05% Tween 20 (24). Signals were revealed using an enhanced chemiluminescence reagent (Kangwei Century Biotechnology Co., Ltd.) and an autoradiography system (CLINX, Chemiluminescence Imaging System).
Rip140
Manufactured by Santa Cruz Biotechnology
Sourced in United States
RIP140 is a protein that functions as a transcriptional coregulator. It can interact with various nuclear receptors and modulate their transcriptional activity.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (2 mentions)
Immobilon p, by Merck Group (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Merck Group (1 mentions)
Atf7ip, by Merck Group (1 mentions)
Tgfbr2, by Santa Cruz Biotechnology (1 mentions)
Phospho p44 42 mapk, by Santa Cruz Biotechnology (1 mentions)
Phospho p38mapk, by Santa Cruz Biotechnology (1 mentions)
P44 42 mapk, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Roche (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
3 protocols using rip140
1
Western Blot Analysis of RIP140 Protein
2
Western Blot Analysis of Protein Markers
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Protein lysates were subjected to 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Membranes were blocked with tris-buffered saline Tween 20 (tris-buffered saline with Tween [TBST]; 20 mmol/L tris-HCl, pH 7.5, 137 mmol/L NaCl, 0.05% Tween 20) containing 5% nonfat milk powder, for 1 hour at room temperature, and incubated with antibodies against RIP140 (1:200; Santa Cruz, CA, USA), Tuj1 (1:500; Sigma, St. Louis, MO, USA), ERK1/2 (1:1000; Cell Signaling Technology, Bilerica, MA, USA), p-ERK1/2 (1:500; Cell Signaling Technology) and β-actin (1:1500; Proteintech Group, Chicago, IL, USA) overnight at 4°C. After TBST washing, blots were incubated with horseradish peroxidase-linked goat anti-rabbit or anti-mouse immunoglobulin G (IgG) antibodies (1:2000; Cell Signaling Technology, USA) for 1 hour at room temperature. Bands were then detected and visualized using epichemilumenescence kits.
3
Western Blot Protein Expression Analysis
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Cells/tissue were harvested, washed in PBS, and resuspended in RIPA buffer (CST-9806) containing proteinase inhibitors (Roche) and quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher). Protein lysates diluted in 4 X Laemmli Sample Buffer (Bio-Rad 161–0747) were loaded onto Bio-Rad 4–20% precast gels. Following electrophoresis, proteins were transferred to a pre-activated PVDF membrane using the Trans-Blot®Turbo™ Transfer System and visualized using ECL (Bio-Rad Chemidoc). Antibodies used in this study are DUSP4 (CST#5149), Cyclin D1 (CST#2978), Cyclin E1 CST#4129), ATF7IP (Sigma#16,578), ATF7IP (Sigma#HPA023505), ADCY3 (Abcam#ab199157), ATXN7 (Invitrogen#PAI-749), CREBBP (CST#7389), SAPK/JNK (CST#9252), LPAR2 (Abcam#ab135980), NF1 (Bethyl#A300-140A-M), Phospho-p38 MAPK (Thr180/Tyr182) (CST#4511), p38 MAPK (D13E1) (CST#8690), p44/42 MAPK (Erk1/2) (CST#4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST#4370), Phospho-SAPK/JNK (Thr183/Tyr185) (CST#4668), TRAIL (CST#3219), RIP140 (Santa Cruz#sc518071), GAPDH (Santa Cruz#sc32233), c-Myc (CST#5605), TGFBR2 (Santa Cruz#sc17792), CASZ1 (Santa Cruz#sc398303), and CFL1 (Abcam#ab42824). Full blots are shown in Additional file 1 : Fig. S11-S13.
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