Western blot
LYSED, SECONDARY
NHBE, PPARα CRISPR-KO NHBE cells, or PPARα-OE HEK293T cells were washed in DPBS, lysed in 1 x Laemmli Loading buffer, and boiled at 100 °C; 40 μl of cleared lysate were analyzed in a pre-cast gradient polyacrylamide gel (Bolt 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel/ NW04120BOX, Invitrogen) using SeeBlue Plus2 Pre-stained Protein Standard (LC5925, Invitrogen) in MES SDS running buffer (B0002, Invitrogen) according to manufacturer’s instructions. The proteins were transferred to a PVDF membrane (iBlot 2 Transfer Stacks, PVDF, mini/ IB24002, Invitrogen) using iBlot2 (LifeSciences). The membrane was blocked with 5% BSA (160069, MPBio) in Tris-buffered saline plus 0.1% Tween 20 (TBST) for 1  hr at room temperature. The membranes were incubated in primary antibodies overnight at 4  °C. The next day, the membranes were washed in TBST (3 × 10  min) and then incubated with horseradish peroxidase-conjugated secondary antibody for 2  hr at room temperature. After the TBST washes (4 × 10  min), EZ-ECL kit (Sartorius; 20-500-1000A, 20-500-1000B) was used to detect the HRP activity. The membrane was imaged on a Vilber Fusion FX and band densitometry was performed on FIJI.The following commercial primary antibodies were used: anti-PPARα (1:1000;ab24509, Abcam) and anti-α-tubulin (1:2000; T6074, Sigma). Commercial horseradish peroxidase-­conjugated secondary antibodies were: anti-rabbit (111-035-003, Jackson) and anti-mouse (115-035-003, Jackson). All primary antibodies were used in 5% BSA in TBST. Secondary antibodies were used at a 1:8000 dilution in TBST.The gel, ladder, and equipment to run and transfer the gel were kindly provided by Prof. Eran Meshorer, Institute of Life Sciences, The Hebrew University of Jerusalem. The anti-tubulin and both HRP-conjugated antibodies, as well as the HRP detection kit, were kindly provided by Prof. Benjamin Aroeti, Institute of Life Sciences, The Hebrew University of Jerusalem.