Mouse anti human synaptopodin

The following antibodies were employed in this study: rabbit anti‐human TRPC6 (Alomone Labs, Jerusalem, Israel); rabbit anti‐human podocin (Abcam, Cambridge, UK) for immunocytochemistry and another one from Santa Cruz (Heidelberg, Germany) for Western blotting; mouse anti‐human synaptopodin (Progen, Heidelberg, Germany); rabbit anti‐human PKCα, rabbit anti‐human PKCδ and rabbit anti‐human PKCζ (Biomol Res Lab, Plymouth Meeting, PA, USA); rabbit anti‐human PKCλ/ι (Santa Cruz); rabbit anti‐human PKCβ₁, rabbit anti‐human PKCβ₂, rabbit anti‐human PKCγ, rabbit anti‐human PKCε, rabbit anti‐human PKCη, rabbit anti‐human PKCθ and rabbit anti‐human β‐actin (Sigma‐Aldrich).

AB8/13 cells, differentiated for 15 days at 37°C, treated with TNFα (10 ng/ml) for 4, 8 and 24 h were lysed by sonication in Radio-Immuno Precipitation Assay (RIPA) buffer (Sigma-Aldrich) for 1 min. Total protein concentration of the lysate was determined by BCA protein assay (Thermo Scientific) according to manufacturer’s protocol and equal amounts of lysate were mixed with SDS-loading buffer. The protein samples were electrophoresed on a SDS-(10%) PAA gel and transferred to a nitrocellulose membrane followed by blocking with 5% skimmed milk in PBS for 1 h. The membrane was cut based on the molecular masses of the target proteins followed by incubation with primary antibodies rabbit- polyclonal anti-VCAM-1 (~100-kDa, Santa Cruz Biotechnology Inc. Heidelberg, Germany), mouse-anti-human synaptopodin (~74-kDa, Progen), rabbit-polyclonal anti-GAPDH (~37-kDa, Santa Cruz Biotechnology Inc.) and/or mouse anti-human actin (~42-kDa, Millipore, Amsterdam, the Netherlands) for 1 h at room temperature. The membranes were washed three times with PBST and incubated with relevant horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Finally, the membranes were washed and the proteins on the membrane were visualized and detected using Supersignal West Femto Chemiluminescent Substrate (Thermo Scientific) and a Gel Doc imaging system equipped with a XRS camera (Bio-Rad).