Sc 1615

Cells were lysed by adding 300 μl of lysis buffer (0.5% IGEPAL-CA-630 Sigma, 0.5 mM EDTA, 20 mM Tris-Base pH 7.6, 100 mM NaCl, protease inhibitor cocktail) into 10 cm plates. All plates were scraped and transferred into 1.5 ml vial on ice and sonicated in an ice bath for two cycles 10 sec, amplitude 30% on the Vibra-Cell Sonics system VCX-750 (Four-element probe, 3 mm stepped microtip). All vials were centrifuged at 10,000 RPM for 10 min at 4 °C. The protein concentration was determined using Bio-Rad DC Protein assay. The normalized and denatured protein samples (containing Laemmli buffer, heated at 70 °C 10 min) were loaded on Precast Bio-Rad Mini-TGX 4–15% polyacrylamide gel (456–1084). The following antibodies were used for immunoblotting: AHR (BML-SA210-0100; ENZO Lifesciences), AHR (sc-5579; Santa Cruz), Actin (sc-1615; Santa Cruz), CYP1A1 (sc-393979; Santa Cruz), and VDR (sc-13133; Santa Cruz). Full-sized blots for all westerns shown in the figures are presented in Figs S5 and S6.

One-week post-transduction, cells were lysed by addition of TRIzol reagent (Invitrogen) and RNA extracted according to the manufacturer’s instructions. After DNase treatment (Invitrogen), RNA was reverse transcribed into cDNA as already described [44 (link)]. Q-PCR reactions were carried out as previously described [45 (link)], using the following primers:
AKTIP Forward 5’- TCCACGCTTGGTGTTCGAT-3’;
AKTIP Reverse 5’-TCACCTGAGGTGGGATCAACT-3’;
TRF2 Forward 5’- TCCTCACGATGGCCAAAAAG-3’;
TRF2 Reverse 5’- GCTGTTTATCTTCCTTCCCTGTACT-3’;
GAPDH Forward 5’-TGGGCTACACTGAGCACCAG-3’;
GAPDH Reverse 5’-GGGTGTCGCTGTTGAAGTCA-3’
and analyzed with the 2^–ΔΔCq method as previously described [46 (link)]. For Western blotting, one week post-transduction with LV-shAKTIP or LV-shctr and 72hrs post-transfection with siRNAs, protein extracts were obtained as previously described [21 (link)] and quantified by Bradford assay. 100μg protein extracts were loaded onto pre-cast 4–12% gradient acrylamide gels (Novex, Life Technology). After electro-blotting filters were incubated with anti-AKTIP (HPA041794 Sigma) and anti-actin-HRP conjugated (sc-1615, Santa Cruz Biotechnology) antibodies. Filters were then incubated with anti rabbit HRP-conjugated secondary antibody (sc-2357, Santa Cruz Biotechnology). Detection was performed using the enhanced chemiluminescence system (Clarity ECL, Biorad).