Dual color protein loading buffer

Protein samples were extracted from HUVEC, HUVEC-PGI2S and HUVEC-PGI2S-tPA cells using ProteoJET Mammalian Cell Lysis Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction, mixed with DualColor Protein Loading Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and boiled for 5 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 4% spacer and 10% separation gels; 30 μg protein was loaded into each well. Afterwards, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA), blocked with 5% non-fat dry milk for 1 h at room temperature, then incubated with primary antibodies specific for PGI2S (52 kDa; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), tPA (65 kDa; 1:500; Bioworld, St. Louis Park, CA, USA), PKA (43 kDa; 1:500; Bioworld), PKC (80 kDa; 1:2000; Epitomics, Burlingame, CA, USA) and MAPK (44 kDa; 1:1000; Cell Signaling Technology, Danvers, MA, USA) or β-actin (45 kDa; 1:1000; Cell Signaling Technology) for 12 h at 4 °C. Membranes were incubated with secondary antibodies labeled with horseradish peroxidase for 1 h, imaged using an ECL system (Pierce, Madison, WI, USA) and densitometric analyses was performed by normalization against the internal loading control β-actin using Image J software (NIH, Bethesda, MD, USA).

Proteins from clinical samples and cell lines were extracted using RIPA lysis buffer. Protein samples were treated with Dual Color Protein Loading Buffer (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated using SDS-PAGE gels (10%) and then transferred to nitrocellulose membranes (NC) (Merck KGaA, Darmstadt, Germany). The membranes were then blocked with Protein-Free Rapid Blocking Buffer (Thermo Fisher Scientific) and incubated at 4 °C overnight with primary antibodies against CDK6 (1:1000) and β-tubulin (1:1000) (Abcam, Cambridge, UK). The next day, the membranes were washed with 1 × TBST three times (10 min each). Then, the membranes were incubated at normal temperature for 1.5 h with a matched secondary antibody (HRP-labeled Goat Anti-Human IgG [H+L], Beyotime Biotechnology, Shanghai, China). Finally, the membranes were exposed to an X-ray film.

Proteins were extracted from tissues and cell lines using RIPA lysis buffer. Following extraction, protein samples were processed with treated with Dual Color Protein Loading Buffer (Thermo Fisher Scientific). Protein separation was achieved through SDS-PAGE using a 10% gel, followed by protein transfer onto nitrocellulose membranes sourced from Merck KGaA (Darmstadt, Germany). The Protein-Free Rapid Blocking Buffer (Thermo Fisher Scientific) was used to block the membranes. The membranes were exposed to primary antibodies against GOLGA8B (dilution 1:1000) and GAPDH (dilution 1:1000; Abcam UK) overnight at 4 ℃. The following day, the membranes were washed thrice using 1 × TBST (10 min/cycle) and then incubated at room temperature for 1.5 h with the corresponding secondary antibody [Catalog No. A0208, HRP-labeled Goat Anti-Rabbit IgG (H + L), obtained from Beyotime Biotechnology, Shanghai, China]. Finally, after exposing the membranes to X-ray film, protein identification was conducted.

Protein was extracted with ProteoJET Mammalian Cell Lysis Reagent (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitor. Protein samples were treated with Dual Color Protein Loading Buffer (Thermo Fisher Scientific) containing reducing agent at 100 °C for 5 min, resolved on 10% Tris-HCl polyacrylamide gels, and transferred to a polyvinylidene fluoride membrane. Overnight incubation (4 °C) of the primary antibody was followed by HRP-conjugated anti-rabbit (1 : 1000; Novus Biologicals, Littleton, CO, USA) or anti-mouse antibody (1 : 1000; Novus Biologicals) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Antibody dilution was as follows: SPAG5, 1 : 300 (Sigma-Aldrich); mTOR, 1 : 1000 (Cell Signaling, Danvers, MA, USA); S6K, 1 : 1000 (Cell Signaling); phosphorylated (pi)-S6K (Thr 389), 1 : 1000 (Cell Signaling); 4E-BP1, 1 : 500 (Cell Signaling); pi-4E-BP1 (Thr 37/46), 1 : 1000 (Cell Signaling); and GAPDH, 1 : 1000 (Abgent, San Diego, CA, USA).

Protein was extracted with RIPA lysis buffer from tissues and cells. Protein samples were treated with Dual Color Protein Loading Buffer [Thermo Fisher Scientific, Waltham, MA, USA, Catalog No. NP0007). SDS-PAGE gels (10% and 15%) were used to separate proteins, followed by transfer to nitrocellulose membranes (Merck KGaA, Darmstadt, Germany, Catalog No. 71078)]. Protein-Free Rapid Blocking Buffer (Thermo Fisher Scientific, Catalog No. 37584) was utilized to block the membranes. Then the membranes were incubated overnight at 4 °C with primary antibodies against c-MYC (1:1000), MYL9 (1:1000), SNAI2 (1:1000) and GAPDH (1:1000). The next day, 1xTBST was used to wash the membranes three times (10 min. each). Then, the membranes were incubated at room temperature for 1 h with a matched secondary antibody (1:1000). Lastly, the membranes were exposed to X-ray film (FluorChem R, Protein Sample, California, USA).