Ab155938

Immunoblotting was done as standard procedures. Cell lysates was separated using SDS-gel electrophoresis and transferred onto PVDF membranes. They were blocked with 5% BSA and incubated with specific antibodies. The following primary antibodies were used: HMGB1 (1:1000; ab5662; Abcam, Cambridge, U.K.), rac1 (1:1000; ab155938; Abcam, Cambridge, U.K.), and cdc42 (1:1000; ab64533; Abcam, Cambridge, U.K.). All Western blots shown were representative results obtained from at least three independent experiments, and all results were analyzed by Image J.

Cells or exosomes were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 1 mMEDTA) supplemented with complete protease inhibitor cocktail (Sigma-Aldrich). Lysates were centrifuged at 12,000 g for 30 min at 4°C to remove insoluble material, after which protein concentrations were determined using a Bradford assay kit using bovine serum albumin (BSA) as a standard according to the manufacturer's instructions. Proteins were resolved on SDS-polyacrylamide gels, and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% (w/v) fat free milk, the membrane was stained with the corresponding primary antibodies followed by incubation with the appropriate secondary HRP-conjugated antibodies. The membrane was incubated with the following primary antibodies: anti-CD63 antibody (Abcam, ab134045), anti-TSG101 antibody (Abcam, ab125011), anti-ALIX antibody (Abcam, ab88388) and anti-CAV1 antibody (Abcam, ab2910), anti-ARF6 antibody (Abcam, ab131261), anti-Rac1 antibody (Abcam, ab155938), anti-CLTC antibody (Abcam, ab21679), anti-GAPDH (Cell signaling).

Transfected cells were cultured for 2 days, then collected and lysed by vortexing with acid‐washed glass balls for 2 min, then placed on ice for 2 min; this was repeated 10 times. The supernatant was gathered after 30 min of centrifugation at 12,000 rpm, and protein concentration measured with NanoDrop (Thermo-Fisher Scientific). Protein (20 μg) was loaded onto 10% SDS-PAGE gels for 1.5 h, then transferred onto polyvinylidene difluoride membranes for 1 h, followed by blocking for 2 h in non-fat milk (5%) in Tris-buffered saline solution containing 0.1% Tween‐20, at normal atmospheric temperature. We then probed the membranes overnight at 4 °C with primary antibodies for Vimentin (1:1000, ab137321, Abcam), GAPDH (1:1000, ab9485, Abcam), N-cadherin (1:1000, ab76057, Abcam), E-cadherin (1:1000, ab231303, Abcam), β-actin (1:1000; sc-47778, Santa Cruz), and RAC1 (1:1000, ab155938, Abcam). We added horseradish peroxidase‐conjugated secondary antibody (anti‐mouse, 1:2000, Santa Cruz) for 2 h at normal atmospheric temperature and visualized protein bands using ECL chemiluminescence detection kit (Thermo-Fisher Scientific).

For western blotting, kidney tissues or podocytes were extracted and quantified. The protein samples were then boiled at 95 °C for 10 min and separated on a 6–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then incubated with primary antibodies overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for protein expression visualization. Primary antibodies were used as follows: NPHS2 antibody (1:500, 20384-1-AP, Proteintech), phosphorylated Nephrin antibody (1:10000, ab80299, Abcam), Active Caspase-3 antibody (1:500, A11021, ABclonal), WT1 antibody (1:1000, A2446, ABclonal), β-actin antibody (1:3000, AB0035, Abways), Bax antibody (1:500, 380709, ZENBIO), RAC1 antibody (1:1000, ab155938, Abcam), β-tubulin antibody (1:3000, AB0039, Abways), OLR1 antibody (1:500, 11837-1-AP, Proteintech), SR-A1 antibody (1:500, 382017, Zenbio), CD36 antibody (1:500, 381350, Zenbio), IGF-1R antibody (1:50, sc-81464, Santa Cruz).