Anti vimentin sc 6260

Anti-phospho (p) Rb at Ser807/811 (catalog number #9308) and anti-Slug antibodies (#9585) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-EBP50 (ab109430), Anti-cyclin A2 (#ab181591), anti-Snail (#ab180714), and anti-Twist1 (#ab50887) antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-E-cadherin, anti-Ki-67 (#M7240), anti-p21^waf1 (#M7202), and anti-cyclin D1 antibodies (#M3635) were from Dako (Copenhagen, Denmark). Anti-p27^kip1 (#610242), anti-Rb (#554136), anti-aldehyde dehydrogenase 1 (ALDH1) (#611194), anti-β-catenin (#610154), and anti-N-cadherin antibodies (#610920) were from BD Biosciences (San Jose, CA, USA). Anti-FLAG M2 (#F3165), anti-ZEB1 (#HPA027524), and anti-β-actin antibodies (#HPA025958) were from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-vimentin (sc-6260) and anti-cyclin B1 antibodies (#sc-752) were from Santa Cruz Biotech (Santa Cruz, CA, USA). Anti-E-cadherin antibody (#M106, Takara Bio Inc., Siga, Japan) was purchased from Cell Signaling Technology (Danvers, MA, USA).

Cells were washed twice on ice with PBS before adding protein lysis buffer (1× protease inhibitor cocktail, Roche, Basel, Switzerland, 1.5mM EDTA, 1mM DTT, 10% glycerol, 25mM HEPES, pH 7.6). The protein concentration was determined by the Bradford assay (BioRad, Hercules, CA) using BSA as a standard. Total protein (20μg) was resolved by 10% SDS-PAGE for subsequent western blot analysis using antibodies against the following proteins (diluted in TTBS (Tween–Tris Buffered Saline: 0.02% Tween-20 in 100mM Tris-CL [pH 7.5], 1:1000) as indicated): anti-p53 (DO7, DAKO, Glostrup, Denmark), anti-NKX2-1 (sc-53136, Santa Cruz, Santa Cruz, CA), anti-Sp1 (sc-14027, Santa Cruz, CA), anti-p65 (sc-8008, Santa Cruz, CA), anti-Snail (sc-28199, Santa Cruz, CA), anti –NF-Y (sc-17753, Santa Cruz, CA), anti-vimentin (sc-6260, Santa Cruz, CA), anti-IKKβ (sc-271782, Santa Cruz, CA), ant-α-tubulin (sc-5266, Santa Cruz, CA), anti-p21 (sc- 6246, Santa Cruz, CA) and anti-E-cadherin (cat. 610182, BD Biosciences, Franklin Lakes, NJ). The gel was transferred to a Hybond-C Extra membrane (GE Healthcare, Little Chalfont, UK) and immunoblotted with primary antibody, as indicated in the figure legends. Anti-mouse or rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody for detection using an ECL western blot detection system.

Western blot analyses were performed as described previously^{43 (link)}. Samples, which were lysed in buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2.5 mM EDTA, 0.125% Nonidet P-40 (v/v), and protease inhibitors, were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were probed with anti-β-catenin (610154; BD Biosciences, San Jose, CA, USA), anti-TGF-β2 (ab36495; Abcam, Cambridge, MA, USA), anti-E-cadherin (610181; BD Biosciences, San Jose, CA, USA), anti-vimentin (SC-6260; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-actin (SC-1615; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. Immunoreactivity was visualized by autoradiography.

Spinal cords were collected on day 1 and 4 from rats implanted with MSC scaffolds and cells treated with recombined human IL-10 (rhIL-10) for 8 h. Total protein was quantification with BCA after lysis in lysis buffer. Proteins were separated by 8-12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. 5% fat-free milk was used for blocking, and T-PBS was used for washing three times. Then, the membranes were cut with molecular weight and incubated with primary antibody. The following primary antibodies were used: Cell Signaling Technology: anti-p-NF-κB (#3033), anti-JAK2 (#3230), anti-p-JAK2 (Tyr1007/1008) (#3771), anti-STAT3 (#9139,), anti-p-STAT3 (Y705) (#9145); Huabio: anti-NF-κB (ET1603-12,), anti-CCR7 (ET1602-22); DiagBio Technology.: anti-GAPDH (db106, 1:5000); Santa Cruz biotechnology: anti-Vimentin (sc-6260). Horseradish peroxidase (HRP)-conjugated IgG (MULTI Sciences) was used to bind the primary antibody for 90 mins at room temperature, and enhanced chemiluminescence (ECL, PerkinElmer) was added to the strip to detect the target protein; images were captured with a digital imager (azure, c280). GAPDH was used as a loading control.

The cells were lysed in RIPA buffer as described previously [4 (link)]. Approximately 20–80 μg aliquots of protein were subjected to SDS–polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were incubated with anti-p-AKT (9271, 1:1000, Cell Signaling, Danvers, MA, USA), anti-AKT (sc-8312, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-catenin (sc-7199, 1:2000, Santa Cruz Biotechnology), anti-Snail (3895, 1:1000, Cell Signaling), anti-vimentin (sc-6260, 1:1000, Santa Cruz), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich) antibodies, and the bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) Western blotting detection reagent (170-5061, Bio-Rad Laboratories, Hercules, CA, USA).

Anti-β-catenin (sc-7963), anti-E-cadherin (sc-8426), and anti-Vimentin (sc-6260) antibodies were purchased from Santa Cruz (Dallas, TX, USA). Anti-GSK-3β (#12456), anti-p-GSK-3β (#9322), anti-AKT (#4691), anti-p-AKT (#4060), and anti-p-β-catenin (#9561) antibodies were purchased from Cell Signaling Technology (California, USA). Anti-HORMAD1 (67091-1-Ig), anti-ZEB-1 (21544-1-AP), anti-LaminB1 (12987-1-AP), and anti-N-cadherin (22018-1-AP) antibodies were purchased from Proteintech (Wuhan, China). Anti-α-tubulin (A01410-100) antibody was purchased from GenScript (Nanjing, China). The Wnt activator (CHIR99021), Wnt inhibitor (XAV939) and AKT inhibitor (MK-2206) were purchased from Selleckchem Chemicals (Houston, TX, USA).