Tubulin t 9026

Whole-cell extracts were generated as described previously^{52 (link)}. In brief, cell monolayers were washed with ice-cold phosphate-buffered saline and then lysed in extraction buffer (1% (v/v) Triton X-100, 10 mm Tris-HCl, pH 7.4, 5 mm EDTA, 50 mm NaCl, 50 mm sodium fluoride, 2 mm Na₃VO₄, containing Complete™ inhibitor mix (Roche Applied Science) per 10 ml of buffer) and homogenised by passage through a 26-gauge needle six times. The lysate was incubated on ice for 10 min prior to centrifugation (14,000 rpm at 4 °C). Equal amounts of protein were resolved by SDS-PAGE using 4–12% gradient gel (Biorad) and then subjected to immunoblot analysis. Antigen-antibody interactions were detected with ECL-reagent (Amersham, UK). Proteins were detected using the following antibodies: ER (ERα(F-10) sc-8002, Santa Cruz Biotechnology) at 1:200, cyclin D1 (CST-2922, Cell Signalling Technology) at 1:1000, PGR (NCL-L-PGR, Novocastra) at 1:500, FOXA1 (Abcam; ab 5089) at 1:1000, GAPDH [6C5] (Abcam; ab 8245) at 1:2000 and Tubulin (T-9026, Sigma- Aldrich) at 1:5000. Uncropped immunoblots are show in Supplemental Fig. 7.

Dulbecco's Modified Eagle's Medium (DMEM), FBS, Penicillin/Streptomycin and Glutamine were all purchased from Life Technologies. BIRB796 (S1574), CB1954 (S7829), PLX-4072 (S1267), Vemurafenib (S1267) and ZSTK474 (S1072) were purchased from Selleckchem; Trametinib (HY-10999) was from Insight Biotechnologies; JNK inhibitor VIII (CAS 894804-07-0) was from Calbiochem; SCH772984 (S7101) was from Merck. Bovine serum albumin (BSA), DMSO and Doxycycline were purchased from Sigma. Vectashield (H-1200-10) was purchased from VectorLabs. The following antibodies were all purchased from Cell Signalling Technologies: p-S473 AKT (4060), Akt1 (2967), c-Jun (9165), p-T202/Y204 ERK1/2 (4370), ERK1/2 (9107), GFP (2955), p-S217/S221 MEK1/2 (9154), MEK1/2 (4694), p-T334 MAPKAPK2 (8753), MAPKAPK2 (3042). BRAF (3967S) was purchased from Santa Cruz. Tubulin (T9026) and β-actin (A5441) were purchased from Sigma and KRAS (12063-1-AP) was from ProteinTech. Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad and the enhanced chemiluminescence (ECL) system from GE Healthcare was used for detection. The Dylight™680 and Dylight™800 conjugated secondary antibodies were from Cell Signalling Technology.

The following antibodies (Ab) were used: ADAM10 purchased from Abcam ab39153 (Cambridge, UK), SAP97 from Stressgen ADI-VAM-PS005-D (San Diego, CA, USA), β2-Adaptin from BD Bioscience 610382 (NJ, USA), Tubulin T9026 and GluN2A M264 from Sigma-Aldrich, GluA1 75-327, PSD-95 75-028, GluN2B 75-097, and GFP 75-132 from Neuromab (Davis, CA, USA), GluA1-p845 04-1073 and 6E10 SIG39320-200 (Covance) from Millipore (Billenca, MA, USA), and GluN1 320500 from Thermo Fisher (Waltham, MA, USA). Peroxidase-conjugated secondary Abs were purchased from Bio-Rad (Hercules, CA, USA). AlexaFluor secondary Abs were purchased from Thermo Fisher.