C digit chemiluminescent scanner

For Southern blot analysis of P. tricornutum genomic DNA from lines containing the p0521s plasmid, DNA was extracted using a modified CTAB protocol12 (link) from P. tricornutum cultures grown on L1 agar containing phleomycin (20 μg ml⁻¹). DNA (∼30 μg) was digested with ClaI that cuts a single time within the p0521s plasmid and still cuts frequently within the P. tricornutum genome. Plasmid control DNA from E. coli was digested with RsrII that also cuts a single time within the p0521s sequence but is not affected by Dam methylation. Digested DNA was separated by agarose gel electrophoresis overnight at 0.1 V cm⁻¹ in a 1% gel. Gels were treated for Southern blot by rinsing once in 0.25 M HCl, twice in denaturing solution (1.5 M NaCl, 0.5 M NaOH) and twice in neutralization solution (1.5 M NaCl, 0.5 M Tris, pH 7.0). Overnight transfers onto Hybond N+ membrane (GE) were performed in 20 × saline-sodium citrate (3 M NaCl, 300 mM Na₃C₆H₅O₇). Hybridization was performed using a probe to the ShBle cassette constructed by the DIG PCR Probe Synthesis Kit (Roche) according to the manufacturer's instruction using primers SB2 and 3′ShBle (Supplementary Table 1). Blots were developed using the bioluminescent substrate CDP-star and imaged on the C-DiGit chemiluminescent scanner (Licor).

Immunoprecipitation and western blotting were performed as described previously (40 (link)). Aliquots of MDA-MB-231 lysates were immunoprecipitated by incubation with 2 μg of anti-TRPC6 antibody and 25 μl of protein A-agarose overnight at 4 °C on a rocking platform. Both immunoprecipitated and lysated samples were resolved by 10% SDS-PAGE, and proteins were transferred onto nitrocellulose membranes for subsequent probing. Residual protein binding sites were blocked by incubation for 60 min at room temperature with 10% (w/v) BSA in tris-buffered saline (in mM): 20 Tris base, 137 NaCl, pH 7.6 at 37 °C, with 0.1% Tween 20 (TBST). Immunodetection of TRPC6, PMCA, ubiquitin, and β-actin was achieved by incubation overnight with anti-TRPC6 antibody diluted 1:500 in TBST (10% BSA), for 2 h with anti-PMCA antibody diluted 1:1000 in TBST (10% BSA), overnight with antiubiquitin antibody diluted 1:1000 in TBST (10% BSA), or for 1 h with anti-β-actin antibody diluted 1:2000 in TBST (10% BSA). The primary antibody was removed and blots were washed with TBST. Blots were then incubated for 1 h with horseradish-peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG antibody diluted 1:10,000 in TBST (1% BSA) and then exposed to enhanced chemiluminiscence reagents for 5 min. The density of bands was measured using a C-DiGit Chemiluminescent Scanner (LI-COR Biosciences).