Sds loading buffer

Construct soluble extracts of MSCs and constructs were analysed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to assess gross quantitative/qualitative differences in protein profiles [20 (link)]. Additionally, Rapigest™ extracts of mesenchymal stems cells prior to differentiation were also evaluated. Then 30 μg was loaded according to equal volumes after ethanol precipitation and resolubilisation in SDS loading buffer (Invitrogen) and stained with Coomassie.

Cells treated with or without GSI at various doses were lysed in Lysis buffer (20 mM Tris pH 7.5/150 mM NaCl/12 mM EDTA/10% glycerol/1% Triton X-100) containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and PhosphoStop (Roche Applied Science, Indianapolis, IN). The protein concentration was measured using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Fifty μg of protein was mixed with an equal volume of 2x SDS loading buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min before applying the sample to SDS-PAGE. After SDS-PAGE, the protein gel was blotted on a nitrocellulose membrane Hybond C extra (Amersham Biosciences, Pittsburgh, PA) using a Bio-Rad blotting apparatus (BioRad, Hercules, CA) for one hour. The blot was blocked with 5% dry milk in TBST for one hour, washed twice in TBST, and then incubated with antibodies (1∶1000) in 5% BSA at 4°C overnight. The blot was washed three times in TBST and then incubated with peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) in 5% dry milk at room temperature for 1 hour. After washing three times in TBST, the blot was incubated for 5 minutes with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and X-ray film (Denville Scientific, Metuchen, NJ) was then developed.

Western blot was performed as described previously^{69 (link)} with minor modifications. Briefly, protein samples were heated at 95 °C for 5 min with 2xSDS loading buffer (Invitrogen) and resolved on 12% SDS-PAGE (Invitrogen). Following electrophoresis, the proteins were transferred to PVDF membrane 0.2 µm (Millipore). Primary mouse anti-GFP antibodies (dilution WB 1:1000) (#11814460001, Roche) and secondary goat anti-mouse IgG (H + L) conjugated with HRP (dilution WB 1:5000) (Cat. No. G-21040, Life Technologies) were used for immunoblotting. Bound primary antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies and the Super Signal West Dura ECL detection reagent (Life Technologies). The specific protein bands were visualized using the Super Signal West Dura ECL detection reagent (Life Technologies) and imaged using the ChemiDoc Imaging System (Bio-rad, Gladesville, New South Wales, Australia).

The cells were transfected with plasmids with Flag label. Afterward, cells were lysed with IP cell lysis buffer (Beyotime) at 4 °C for 30 min. Cell lysates were then incubated with anti-Flag antibody (Sigma-Aldrich) overnight at 4 °C. Protein A/G agarose beads (Santa Cruz Biotechnology) were added to the samples and incubated at 4 °C for 4 h. The immune complexes were subsequently washed three times with PBS and eluted with 2× SDS loading buffer (Invitrogen). The eluates were subjected to western blot analysis using the indicated antibodies. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed by Wininnovate Bio (Shenzhen, China).

The in vitro methyaltion assays were carried out as described previously (31 (link),33 (link),34 (link)). Briefly, PRMT5 was immunoprecipitated and incubated with recombinant His-TDP1^WT or His-TDP1^KK proteins (1.5 μg). Methylation reactions were carried out using methylation buffer (50 mM Tris–HCl, (pH 8.5), 5 mM MgCl₂, 4 mM DTT) containing 100 μM unlabeled S-(5′-adenosyl)-l-methionine chloride dihydrochloride (SAM) (A7007) (Sigma) for 2 hours at 30°C. Reactions were stopped by adding 2× SDS loading buffer (Invitrogen) and boiling the samples for 5 minutes. Methylation reaction products were separated by SDS–PAGE, transferred on to PVDF membrane and analyzed by Western blotting using anti-SDMA and anti-TDP1 antibodies.