Gtx121937

Total cell proteins were prepared from the DM1 model cells, as previously described (Nakamori et al., 2017 (link)). Then, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the following primary antibodies: mouse anti-IGFBP3 (1:500; MAB305, R&D Systems), rabbit anti-PAI-1 (1:2000; NBP1-19773, Novus), rabbit anti-AKT (1:500; GTX121937, GeneTex), rabbit anti-phospho-Akt (Ser473) (1:500; 4,058, Cell Signaling Technology), rabbit anti-p53 (1:100; 2,527, Cell Signaling Technology), rabbit anti-p21 (1:1,000; 2,947, Cell Signaling Technology), rabbit anti-p16 (1:1,000; ab108349, Abcam), and rabbit anti-GAPDH (1:1,000; G9545, Sigma-Aldrich). After incubation, the immunoblots were washed, incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin (Ig) G or anti-rabbit IgG (GE Healthcare), and detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare) using a ChemiDoc Touch Imaging System (Bio-Rad).

For Western blotting analysis, antibodies against AKT (GTX121937), and GAPDH (GTX100118) were purchased from GeneTex (Hsinchu, Taiwan); anti-pAKT^S473 (CST4060), anti-GSK-3β (CST9315), and anti-pGSK-3β^S9 (CST9323) were purchased from Cell Signaling (Danvers, MA, USA). The PI3K inhibitor LY294002 and Akt inhibitor SH-6 were obtained from Enzo Life Sciences (Farmingdale, NY, USA). CA strains SC5314 and SC5314-GFP were gifts from Dr. Lo Hsiu-Jung (National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Taiwan). A plasmid containing cDNA encoding a GSK-3β mutant GSK-3β^S9A was a gift from Scott Friedman lab (Addgene plasmid # 49492; http://n2t.net/addgene:49492; RRID:Addgene_49492). The cDNAs was subcloned into pLentiviral vector to generate lentivirus carrying GSK-3β^S9A cDNA. Lentiviral pGIPZ carrying either shRNA vectors targeting human LDOC1 (shLDOC1) or GFP-tagged LDOC1 ORF were purchased from GE Dharmacon (Lafayette, CO, USA).

Primary antibodies against focal adhesion kinase (FAK; #3285s), phospho-FAK (#8556s), and the mechanistic target of rapamycin (mTOR; #2972s) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against AKT (#GTX121937), phospho-AKT (#ab28821), PDGFRB (#GTX61115), and phospho-PDGFRB (#GTX61797) were purchased from GeneTex (Irvine, CA, USA). Primary antibodies against cyclooxygenase-2 (COX-2; #160126), inducible nitric oxide synthase (iNOS; #610432), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #NB300-221), and phospho-mTOR (#ab109268) were purchased from Cayman Chemical (Ann Arbor, MI, USA), BD Transduction Laboratories (Lexington, KY, USA), Novus Biologicals (Centennial, CO, USA), and Abcam (Cambridge, MA, USA), respectively. Horseradish peroxidase- (HRP-) conjugated secondary antibodies against mouse (#GTX213112-01) and rabbit (#GTX213110-01) immunoglobulin G were also purchased from GeneTex. All other reagents that are not listed were purchased from Sigma-Aldrich (Louis, MO, USA).

Freshly obtained skin samples from mice back with hair removal were prepared in a lysis buffer containing 1% Triton X-100, 1% deoxycholic acid, 2mM CaCl₂ and protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, 1.8mg/ml iodoacetamide and 1mmol/l phenylmethyl sulfonyl fluoride) and quantified with a BCA protein assay kit (Pierce). Equal amounts of total protein were subjected to electrophoresis on 12% Bis-Tris gels, transblotted onto nitrocellulose membranes and probed with different primary antibodies: Anti-p53 (1:1000, sc-393031,Santa Cruz), anti-p16 (1:750, sc-1681,Santa Cruz), anti-p21 (1:250, sc-397,Santa Cruz),anti-GAPDH (1:1000, HC301-01, Transgen Biotech), anti-Akt (phosphoSer473, 1:5000, GTX28932,GeneTex), anti-Akt (Akt 1+2+3)(1:1500, GTX121937,GeneTex), anti-NF-κB (1:500, sc-8008,Santa Cruz), anti-NF-κB (phosphor Ser536,1:1000,93H1, Cell Signaling Technology), anti-Pten (phosphoThr366/pS370, 1:2000, GTX54620, GeneTex), anti-Pten (1:1000, 138G6,Cell Signaling Technology) respectively, followed by a peroxidase-conjugated secondary antibody (KPL). Immunoreactive bands were detected using ECL kit according to the manufacturer’s instructions. Subsequent reprobing using anti-GAPDH was performed for internal loading control.

Western blotting was performed as previously described [27 (link)]. RIPA lysis buffer with 1 mM protein phosphatase inhibitor was added into cells for protein extraction. The lysate was collected and centrifuged at 12,000 r/min for 10 min at 4 ℃. The BCA protein assay kit (Thermo Scientific) was used to determine the concentration of proteins. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% BSA (Sigma-Aldrich, Darmstadt, Germany)-TBST buffer solution (Bio Froxx, Guangzhou, China) for 1 h at room temperature, incubated with antibodies against SFPQ (ab38148, Abcam, U.K.), AKT (GTX121937, GeneTex, USA), p-AKT (WLP001a,Shenyang Wanlei Biotechnology Co., Ltd., China), RUNX2 (ab23981, Abcam, U.K.), and β-actin (30101ES, Shanghai Yisheng Biological Co., Ltd., China) at 4 ℃ overnight. Subsequently, incubated with horseradish peroxidase-conjugated secondary antibodies (SE134, SA131, Solarbio, China) for 1 h. Protein bands were detected using a chemiluminescence system (Bio-Rad Laboratories, Hercules, CA, USA) with an enhanced chemiluminescence (ECL) kit (Millipore, Darmstadt, Germany). Signal intensity was compared using the Image J software (NIH, Bethesda, MD, USA).

Whole protein extracts of the cells for western blotting were prepared using PRO-PREP lysis buffer (Intron, #17081), and protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, #23227). Proteins were separated by 10% SDS-PAGE, blotted onto nitrocellulose membranes, and then probed with antibodies against total AKT (Genetex, #GTX121937, 1:3000 dilution), phosphorylated AKT (Genetex #GTX61708, 1:2000 dilution), total PTEN (Genetex, #GTX101025, 1:500 dilution), and phosphorylated PTEN (Genetex, #GTX61780, 1:1000 dilution). The membranes were then incubated with a goat anti-rabbit IgG secondary antibody (Jackson, #003318367, 1:4000 dilution) for 1 hour. The membranes were incubated in ECL-prime solution (GE Healthcare Amersham, #RPN2232) in the dark for 1 minute and then exposed under a fluorchemHD2 (Cell biosciences) for visualization.