α tubulin sc 32293

Whole cell extracts were obtained using RIPA lysis buffer (VWR Life Science, Radnor, PA, USA, cat# N653-100ML) supplemented with 1× protease inhibitor and 100 mM of phenyl methylsulfonyl fluoride (PMSF) (Amresco, Solon, OH, USA, cat# M145-5G). Protein concentration was quantified via the Bradford Assay using the Bio-Rad Protein Assay Dye Reagent Concentrate (BioRad, Hercules, CA, USA, cat# 500-0205). For western blot analysis, 50 μg of protein were resolved by 15% SDS-PAGE gels and subsequently transferred onto nitrocellulose membranes. Membranes were blocked in 5% BSA or 5% milk in TBS-T for 1 h at room temperature, incubated overnight at 4 °C in primary antibodies. Next, membranes were washed in 1× TBS-T, incubated with secondary antibodies for 2 h, washes with 1× TBS-T and imaged using the Odyssey CLx imager with infrared fluorescence (LI-COR, Lincoln, NE, USA). The primary antibodies used were Claudin 1 (13050-1-AP), Claudin 4 (16195-1-AP) and Claudin 7 (10118-1-AP) (Proteintech, Rosemont, IL, USA), Claudin 3 (341700; Invitrogen, Waltham, MA, USA), GAPDH (5174S; 1:1000; Cell Signaling, Danvers, MA, USA), α-tubulin (sc-32293; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies used were anti-mouse (925-32210; 1:15,000; LI-COR) and anti-rabbit (925-32211; 1:15,000; LI-COR). Original blots can be found at Figures S1 and S2.

Immunoblot analysis was performed as previously described [19 (link)] with primary antibodies to phosphorylated Akt (#4060), to Akt (#9272), to phosphorylated ERK1/2 (#4370), to ERK1/2 (#9102), to phosphorylated MEK (#9121), to MEK (#9122), to phosphorylated S6 (#4858), to S6 (#2217), to phosphorylated STAT3 (#9131), to STAT3 (#4904), and to SHP-2 (#3752), all of which were obtained from Cell Signaling Technology, as well as with those to BRAP (sc-166012), to neurofibromin (sc-376886), and to α-tubulin (sc-32293) from Santa Cruz Biotechnology. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare and Dako), enhanced chemiluminescence reagents (ImmunoStar LD, Wako), and a LAS-3000mini instrument (GE Healthcare, Chicago, IL, USA). After the detection of the phosphorylated forms of Akt, ERK1/2, MEK, S6, and STAT3, the membrane was stripped with WB Stripping Solution (Nacalai Tesque) and then reprobed with antibodies to the corresponding total forms of these proteins.

Antibodies for survivin (# 2808), BCL-2 (# 2872), XIAP (# 2042), caspase-9 (# 9502), caspase-8 (# 9746), caspase-3 (# 9662), C/EBPα (# 2295), C/EBPβ (# 3087) and PARP (# 9542) were purchased from Cell Signaling; anti-BAX (336400) was supplied by Invitrogen, and anti-GAPDH was provided by Genetex (GTX627408); antibodies for P73 (sc-7957), hnRNPA1 (sc-10029), p-H3S10 (sc-8656-R) and α-tubulin (sc-32293) were obtained from Santa Cruz Biotechnology. The antibody against actin was a generous gift from Dr. M. Hernandez, Cinvestav-IPN, Mexico. Commercial secondary anti-mouse (115-095-008) and anti-rabbit IgG (111-095-003) conjugated with Fluorescein (FITC) were purchased from Jackson Immunoresearch.