Dylight 594 conjugated secondary antibodies

Cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilised with 0.01% Triton X-100 for 15 minutes. The cells were then incubated with the desired primary antibodies at 37°C for 1 hour, followed by species-specific secondary antibodies at 37°C for 1 hour. In the growth kinetics studies, the samples were probed with primary antibodies against Alphaviruses (Santa Cruz sc-58088, 1∶250 dilution), followed by FITC-conjugated secondary antibodies (Millipore, 1∶500 dilution). In the polarized infection studies, the samples were co-labeled with primary antibodies against CHIKV E2 glycoproteins (1∶100 dilution) and FITC-conjugated secondary antibodies (Millipore, 1∶250 dilution) together with primary antibodies against ZO-1 proteins (Invitrogen, 1∶500 dilution) and Dylight-594-conjugated secondary antibodies (Thermo Scientific, 1∶100 dilution). In the virus binding assay, the samples were probed with primary antibodies against Alphaviruses (Santa Cruz, 1∶250 dilution), followed by FITC-conjugated secondary antibodies (Millipore, 1∶500 dilution). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 20 minutes. 1×PBS washes were performed after each incubation step. The samples were subsequently mounted onto glass slides using DABCO and viewed under the Olympus IX81 inverted microscope or Olympus FV1000 confocal microscope.

C3H10T1/2 cells were seeded on coverslips. After different treatment, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in TBS for 15 min. After being blocked in 5% FBS in PBS for 30 min, cells were incubated with anti-β-catenin (Cell Signaling Technology) overnight at 4 °C. After washing three times with PBS, cells were incubated with DyLight™ 594-conjugated secondary antibodies (Thermo Scientific) for 60 min at room temperature and then stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 10 min. Slides were then washed three times and mounted. Immunofluorescence was detected using an Olympus inverted fluorescence microscope.

Antibodies specific for podocin, synaptopodin, vimentin, mmp9, WT1 and phospho‐FAK (phospho Y397) were from Abcam (Cambridge, MA, USA). Anti‐NEPH1 antibodies purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti‐p38 MAPK, phospho‐p38 MAPK (Thr180/Tyr182) and FAK antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti‐β‐actin antibodies were obtained from Proteintech (Chicago, IL, USA). Secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA) and Dylight 594 conjugated secondary antibodies were from Pierce, Thermo Fisher Scientific Inc. (Rockford, IL, USA). mmp9 ELISA kits were from Abcam. The p38 inhibitor SB203580 was from Selleck (Houston, TX, USA). LDL and FITC‐Phalloidin were purchased from Sigma Chemical Company (St Louis, MO, USA). The FAK inhibitor TAE226 was from Novartis Pharmaceuticals (Plantation, FL, USA). For in vitro use, SB203580 was first diluted in DMSO as a stock liquid (50 mM); for further use, the stock liquid was added for a terminal concentration at 5 μg/ml 26. For oxidation, LDL (5 mg/ml) were mixed with 5 μmol CuSO₄, incubated for 18 hrs at 37°C, and oxidation was evaluated as described previously 27.