Cav 2

Cell culture reagents were purchased from Gibco. Antibodies and reagents used were as follows: Cav-2 (BD 610685), lamin A/C (BD 612162) and E-cadherin (BD 610182), antibodies from BD Transduction Laboratories; lamin A/C (sc-20681), lamin B1 (sc-30264), α-tubulin (sc-5286), maltose-binding protein (MBP) (sc-73416), emerin (sc-15378), GFP (sc-9996), histone H3 (sc-10890), histone H1 (sc-8030), protein-tyrosine phosphatase 1B (PTP1B) (sc-1718), GCN5 (sc-20698) and p300 (sc-585) antibodies from Santa Cruz Biotechnology; pY19-Cav-2 (ab3417), lamin B-receptor (LBR) (ab169306), H3K9me3 (ab8898), H3K9ac (ab32129), H3K18ac (ab1191) and H3K27ac (ab4729) antibodies from Abcam; GFP (#2555) antibody from Cell Signaling; AcH3 (06-599), RNA Pol II (05-623), H3K4ac (07-539), and H3K14ac (07-353) antibodies from Millipore; FITC-conjugated anti-mouse (F9006), TRITC-conjugated anti-rabbit (T5268), horseradish peroxidase (HRP)-conjugated anti-mouse (A4416) and anti-rabbit (A6154) antibodies and 4′-6-diamidino-2-phenylindole (DAPI) (D8417), curcumin (C1386), butyrolactone 3 (M2449) and sodium ortho-vanadate (S6508) from Sigma; human insulin from Eli Lilly.

Cells were washed using ice-cold PBS and lysed in 70 μl of 1X Laemmli sample buffer (60 mM Tris-Hcl, pH 6.8, 10% glycerol, 2% SDS). Protein determination was performed using the Biorad DC protein assay and 0.01% bromophenol and 5% β-mercaptoethanol were added. 20μg protein was loaded per lane on TGX 4–15% Criterion gels (Bio-Rad, no. 567-1084). Following 1D separation and transfer to nitrocellulose, proteins were detected using the following antibodies: CAV1 (Cell Signaling, cat. D46G3), CAV2 (BD Transduction Laboratories, 610685), CAV3 (BD Transduction Laboratories 610421), CAVIN11 (Abcam ab48824), CAVIN2 (Abcam, ab113876), CAVIN3 (Proteintech, 16250-1-AP), MKL1/MRTF-A (Cell Signaling Technology, #14760), CNN1 (Abcam, ab46794), HSP90 (BD Transduction Laboratories, 610418). Primary antibodies were used at dilutions recommended by the vendors. Secondary HRP-conjugated anti mouse or anti rabbit antibodies (Cell Signaling) were used for detection with SuperSignal West Pico Chemiluminescent Substrate (Pierce, cat34080) in an Odyssey Fc Imager (LI-COR Biosciences). Image Studio 3.1 was used for image capture and analysis.