α tubulin sc 23948

Cells were lysed with RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, Burlington, MA, USA), and protein concentrations in the extracts were measured using the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by SDS–polyacrylamide gels and transferred onto PVDF membranes (Millipore-Sigma). The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.04% Tween-20 (TBST) and incubated with primary antibodies overnight at 4 °C. Antibodies against Cyclin A (#4656), Cyclin B (#4138), γH2AX (#9718), p-Chk1 (#2348), and p-Chk2 (#2197) were purchased from Cell Signaling Technology (Danvers, MA, USA), while PARP (sc-7150), p53 (SC-126), GAPDH (SC-32233), β-actin (SC-130657) and α-Tubulin (SC-23948) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The antibody against TONSL (ab101898) was purchased from Abcam (Cambridge, UK). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Immuno Research, Philadelphia, PA, USA) for 1 h at room temperature. After washing with TBST, the immunoreactive bands were detected using ECL and visualized on X-ray films or with an Image 680 LAS (GE healthcare, Amersham, UK) imaging system.

The cell lysates were prepared as described previously [20 (link)]. Briefly, the Tris-based buffer containing phosphatase inhibitors and protease inhibitors (Roche, Basel, Switzerland) was applied and cleared with centrifugation. The protein in soluble fractions was quantified using a BCA kit (Thermo) and standard western blotting was performed using PVDF membrane (Merk Millipore, Boston, MA, USA). The antibodies used were as follows: α-tubulin (sc-23948) and PARP1/2 (sc-7150) were purchased from Santa Cruz (Santa Cruz, CA, USA). SCD1 (ab19862) was purchased from Abcam (Cambridge, UK). Cleaved active caspase-3 (#9661) was purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies conjugated with horseradish peroxidase were purchased from Cell Signaling Technology. The signals were visualized using SuperSignal^® Femto (Thermo) and X-ray films (Agfa, Mortsel, Belgium).

Keratinocytes were grown on glass coverslips, fixed with 3,8% formaldehyde in PBS for 10 minutes and permeabilized with cold methanol for 5 minutes or fixed with cold methanol for 10 minutes. Paraffin-embedded skin sections were prepared following convential procedures. Samples were incubated for 1 hour (cells) or overnight (sections) in a wet chamber with antibodies against keratin 10 (sc-23877), phospho-histone H3 (sc-8656-R), cyclin A (sc-56299), cyclin E (sc-198), αTubulin (SC-23948) (all from Santa Cruz, CA, USA), involucrin (I-9018, Sigma-Aldrich, Inc), keratin 1 (PRB-149P, Covance, Vienna, VA, USA) or Ki67 (ab16667, Abcam, Cambridge, UK). Then, primary antibodies were revealed with Alexa Fluor-conjugated goat anti rabbit or anti mouse antibodies (Jackson ImmunoResearch, PA, USA). Samples were then incubated with 0.2 µg/ml DAPI (Vector Lab, Burlingame, CA). Coverslips were mounted with Prolong Gold Antifade Reagent (ThermoFisher Scientific, Waltham, MA) and cells were visualized and photographed with a Zeiss fluorescent microscopy. Immunofluorescence staining was scored by counting 400 cells.

Cells were lysed with RIPA buffer (25 mM Tris, pH7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease inhibitors (Roche, 4693132001) and phosphatase inhibitors (Roche, 4906845001) and sonicated with Bioruptor Plus at High setting. Cell lysates were resuspended in 4× sample buffer (200 mM Tris, pH 6.8; 8% SDS; 40% glycerol; 0.4% bromophenol blue and 20% 2-mercaptoethanol) and heated at 95 ℃ for 5 min. Antibodies used were OPA1 (67589), DRP1 (8570), Mitofusin-1 (14793), Mitofusin-2 (11925), H2AX (2595), γH2AX (2577), H3 (9715), H3K4me1 (5326), H3K4me3 (9751), IRF3 (11904), p-IRF3 (S396) (29047), TBK1 (3504), p-TBK1 (S172) (5483), p65 (8242) and p-p65 (S536) (3033) from Cell Signaling; H3K4me2 (39679), H3K9me1 (39887), H3K9me2 (39683), H3K9me3 (39765), H3K27me1 (61015), H3K27me2 (39919), H3K27me3 (39155), H3K36me1 (61351), H3K36me2 (39255), H3K36me3 (61101) from Active Motif; α tubulin (sc-23948) from Santa Cruz; Lamin A/C (GTX101127) from GeneTex. α tubulin was in 1:20000 dilution. For Lamin A/C and all histone-related antibodies, antibodies were used in 1:5000 dilution. Others were in 1:1000 dilution.

The SPARC (#5420), p-SMAD2 (#3108), p-SMAD3 (#9520), SMAD2 (#3103), SMAD3 (#9523), SMAD4 (#38,454), Snail (#3895), Slug (#9585) antibodies were purchased from Cell Signaling Technology. The aromatase antibody was purchased from Bio-Rad Laboratories (#MCA2077). The VEGF antibody was purchased from Thermo Fisher Scientific (#MA5-13182). The α-tubulin (#sc-23948) antibody was purchased from Santa Cruz Biotechnology. The SPARC (#8725) antibody for immunohistochemistry was purchased from Cell Signaling Technology. The recombinant human TGF-β1 was obtained from R&D systems. The SB431542 was obtained from Sigma.

The p-EGFR (#3777), p-HER2 (#2243), HER2 (#2165), p-HER4 (#4757), HER4 (#4795), p-ERK1/2 (#9106), ERK1/2 (#9102), phospho-AKT (#9271), and AKT (#9272) antibodies were purchased from Cell Signaling Technology. The EGFR (#sc-373746) and α-tubulin (#sc-23948) antibodies were purchased from Santa Cruz Biotechnology. The recombinant human HB-EGF was obtained from R&D systems. The AG1478, AG825, and LY294002 were obtained from Sigma. The U0126 was obtained from Cayman.