After rinsing with pre-cooling PBS, MKN-45 cells were dissociated with RIPA buffer (Thermo Fisher Scientific) mixed with protease and phosphatase inhibitor cocktail (Roche). The total protein of MKN-45 cells was isolated on the basis of instructions. The Bio-Rad assay system (Bio-Rad Laboratories, Hercules, CA, United States) was used to detect the protein concentration. In Western blot analysis, 12% sodium dodecyl sulfate-polyacrylamide gels were prepared and the same amounts of protein extract were separated by electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking in 5% skim milk, the blotted membranes were immersed in primary antibodies (SRSF1, 1:1000; GAPDH, 1:2000, Proteintech, Wuhan, China) for 24 h. The membranes were rinsed with TBST three times (10 min/time). Subsequently, the membranes were immersed in a secondary antibody (HRP conjugated goat anti-rabbit antibody, 1:1000, Sigma) at room temperature for 1 h. GAPDH was used as the internal control. Kodak film (Kodak, Rochester, NY, United States) was used to detect the blots with an enhanced chemiluminescence kit (Merck Millipore).
Srsf1
Manufactured by Proteintech
Sourced in United States
SRSF1 is a protein that functions as a splicing factor, involved in the regulation of alternative splicing of pre-mRNA. It belongs to the serine/arginine-rich (SR) family of splicing factors.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (2 mentions)
β actin, by Proteintech (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti rabbit antibody, by Merck Group (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Mabe50, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ino80, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca assay, by Beyotime (1 mentions)
Pvdf western blotting membrane, by Roche (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Collagen 1, by Affinity Biosciences (1 mentions)
β catenin, by Proteintech (1 mentions)
5 protocols using srsf1
1
Protein Expression Analysis in MKN-45 Cells
2
Comprehensive Western Blot Antibody Protocol
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For Western blots, following antibodies were used: FLAG‐M2 at 1:1,000 dilution (mouse, Sigma F1804), 1:2,500 β‐Actin (mouse, Sigma A1978), 1:1,000 SRSF1 (Rabbit, Proteintech 12929‐2‐AP), 1:1,000 SRSF2 (Rabbit, Proteintech 20371‐1‐AP), 1:1,000 Anti‐Phosphoepitope SR proteins, clone 1H4 mouse (MABE50, Millipore Sigma), GAPDH (mouse, Santa Cruz sc‐32233), 1:1,000 eIF2α/EIF2S1 (phospho S51) (rabbit, Abcam ab32157), and 1:1,000 GFP (mouse, Roche/Sigma 11814460001) in 5% non‐fat dry milk. HRP‐conjugated goat‐anti‐mouse (115‐035‐146) or goat‐anti‐rabbit (111‐035‐144) secondary antibodies (Jackson ImmunoResearch Laboratories) were used at a 1:10,000 dilution in 5% non‐fat dry milk.
3
Protein Analysis of CM Samples
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The total protein was extracted from CMs with RIPA lysis buffer (Beyotime, China), and a BCA assay (Beyotime, China) was performed to quantify the protein concentrations. The equal amount of protein samples was loaded and resolved on SDS-PAGE gel, and transferred to nitrocellulose membrane (Millipore, USA). The membranes were blocked in 5% milk and then reacted overnight with primary antibodies against Ino80, Dis3, Srsf1 and GAPDH (Proteintech, USA) at 4 °C. The membranes were finally incubated with secondary antibodies for 1 h at RT and scanned with Odyssey (LI-COR Biosciences, USA).
4
Western Blot Analysis of Protein Markers
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Cells were lysed by using RIPA lysis buffer (CWBIO, China). After adding phosphatase inhibitor (100×) and Protease Inhibitor Cocktail (100×), the cell lysate underwent ultrasonication. The protein extracts were separated using 12% SDS gel electrophoresis and were then transferred to PVDF Western Blotting Membranes (Roche Diagnostics Gmbh Mannheim, Germany). After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies of SRSF1 (1:1000, Proteintech), β-actin (1:1000, Proteintech), and GAPDH (1:1000, Cell Signaling), and cultured using secondary antibodies. Protein blots were cut prior to hybridisation with antibodies during blotting. Finally, the experimental results were visualized by using SAGECREATION and analyzed by Image J.
5
Western Blot Analysis of Protein Expression
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Protein samples were extracted from cells and tissues using RIPA lysis buffer with protease inhibitor (Beyotime, Jiangsu, China). The proteins were separated on a 10% or 8% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Pall Life Sciences, Ann Arbor, MI, USA). Proteins were identified with antibodies against FN1 (1:500, Proteintech, Wuhan, China), Collagen I (1:500, Affinity Biosciences, OH, USA), SRSF1 (1:500, Proteintech, Wuhan, China), and β-catenin (1:500, Proteintech, Wuhan, China). β-Actin (1:1000, Proteintech, Wuhan, China) served as a loading control. The membranes were incubated with secondary antibodies (1:8000, Abcam, USA) for 1 h at room temperature. Finally, Odyssey (Odyssey CLX, Biosciences, USA) was used to detect and quantify protein expression.
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