Immobilon pvdf membrane

For western blot analysis of biotinylated proteins [80 (link)], 6x protein loading buffer was added to lysates, samples were boiled for 5 minutes, followed by cooling on ice. 15 μl lysate was loaded and run on 10% SDS gel in Tris running buffer by SDS-PAGE. Proteins were transferred from gels onto Immobilon PVDF membrane (Millipore Sigma) with a Pierce G2 fast blotter in Pierce 1-Step transfer buffer, then blocked with 3% BSA in TBST overnight at 4°C. Blots were incubated with streptavidin-HRP in 3% BSA in TBST for 30 minutes at room temperature, washed 4 x 5 minutes in TBST, incubated with chemiluminescent substrate (Li-Cor 92695000, Lincoln, NE) for 5 minutes, and imaged using a C-DiGit blot scanner (Li-Cor). For western blot analysis of RNAi knockdowns, cell pellets were lysed in ice cold RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% triton X-100, pH 7.5) with Halt protease inhibitors (Pierce) for 30 minutes on ice, with vortexing every few minutes. Lysates were centrifuged for 20 minutes at 14,000 x g, and supernatant added to 4x laemmli buffer (Bio-Rad, Hercules, CA). Lysates were run by SDS-PAGE on 5–15% or 5–20% mini-PROTEAN TGX stain free gels (Bio-Rad), transferred to Immobilon PVDF membrane (Millipore Sigma), blocked for 1 hr in 5% milk-TBST, and labeled with antibody and digitally imaged as described above.

Purified CaMKK2 was denatured in SDS-PAGE sample buffer, separated by SDS-PAGE and transferred to Immobilon PVDF membranes (Millipore). Membranes were blocked for 1 hr in PBS/1% Tween-20 (PBS-T) supplemented with 5% non-fat milk. Primary antibodies were diluted in PBS-T containing 1% non-fat milk at the following dilution: mouse or rabbit anti-Flag (Cell Signaling; 100 ng/ml), rabbit anti-pThr85 (300 ng/ml), anti-pSer85 (3000 ng/ml) and mouse anti-pSer antibody (BD Bioscience; 250 ng/ml), which selectively detects phosphorylated Ser129/Ser133 on CaMKK215 (link). After incubation with primary antibody solutions for 1 hr, the membranes were briefly washed in PBS-T, and then incubated with appropriate fluorescently labeled secondary antibodies, either goat anti-mouse IgG IRDye800 or goat anti-rabbit IgG IRDye680. Membranes were then scanned with an Odyssey Infrared Imager (Li-Cor).

Protein concentrations were measured using the Bio-Rad Bradford protein assay. The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis, transferred to IMMOBILON PVDF membranes (Millipore) and incubated overnight at 4°C in blocking buffer containing 2% ECL blocking reagent (GE Healthcare) and 0.1% Tween 20 in PBS. The membranes were probed with either anti TFPI-2 (1:500, SantaCruzBiotechnology) or anti α-tubulin (1:1000, Sigma-Aldrich) antibody for 1h at room temperature. Antibody binding was detected with anti-mouse IgG-HRP (1:10,000, Sigma Aldrich) for 45min at room temperature. The signals were detected with ECL Prime (GE-Healthcare). Densitometry of protein bands was analysed with GelPro Analyzer Software (Media Cybernetics).