T9284

For immunoblotting analysis, BMSCs incubated with 50 ng ml^–1 IL-17 for 15, 30, and 60 min or 10 ng ml^–1 TNFα for 7.5, 15, 30 min were pre-challenged by 100 nM RA for 6 h. Cells were washed with ice-cold PBS, harvested and lysed for 15 min by lysis buffer containing 0.5% TritonX-100 (T9284, Sigma), 20 mM Hepes pH7.4 (H-4034, Sigma), 150 mM NaCl (A100241, Sangon Biotech), 12.5 mM β-glycerophosphate (A500486, Sangon Biotech), 1.5 mM MgCl₂ (M4880, Sigma), 2 mM EGTA (A600077, Sangon Biotech), and a cocktail of protease inhibitors, Na₃VO₄ (A600869, Sangon Biotech), NaF (A500850, Sangon Biotech), and PMSF (A610425, Sangon Biotech). Equal amounts of protein extracts were resolved in 10% SDS-PAGE and transferred to PVDF membranes (IPVH00010, Merck Millipore). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST, pH 7.6) for 1 h at room temperature before incubated overnight with the primary antibodies (p65 1:1,000, pp65 (Ser536) 1:1,000, IκBα 1:1,000, pIκBα (Ser32) 1:1,000, β-actin 1:1,000) at 4°C and then incubated with the secondary antibodies (rabbit, 1:10,000, W401B, Promega) for 1 h at room temperature. Finally, the blots were detected by enhanced chemiluminescent reagents (Millipore).

THP-1 cells were seeded on glass coverslips and differentiated into macrophages overnight, before fixation with 4% formaldehyde (FA; Sigma-Aldrich, F1635) containing 0.2% Triton X-100 (Sigma-Aldrich, T9284) for 10 minutes and permeabilized with 1% Triton X-100 for 10 minutes. Fixed cells were washed with PBS and non-specific binding was blocked with 0.5% milk in PBS before incubation with primary antibodies in 0.25% milk in PBS for 1hr. Unbound antibody was removed by washing with PBS and secondary antibody in 0.25% milk in PBS was added and incubated for 30 minutes. Unbound secondary antibody was removed and cells were washed with PBS and stained with DAPI (Sigma-Aldrich, D9542) for 1 minute. Coverslips were washed and mounted with Mowiol mountant (Sigma-Aldrich, 324590). Fluorescence was visualized with a DeltaVision Spectris Deconvolution Microscope (GE Healthcare Life Sciences) with a 100x PlanApo 1.35 objective (Olympus). Images were deconvolved with the SoftWoRx software (GE Healthcare Life Sciences). For the experiments studying the effect of PPAR ligands on FAMIN expression, the number of peroxisomes was calculated using a custom ImageJ macro on 10 images per condition and triplicate experiments. Values were normalized according to the controls (untreated cells).

Cryosections from embryos collected at E12.5 were prepared and processed for staining as in Charoy et al., 2012 (link). For some experiments, chick embryo sections and open-book spinal cords were blocked in 6% BSA (A7906, Sigma) and 0.5% Triton (T9284, Sigma) diluted in PBS for 5 hr at room temperature. Sections were incubated overnight at room temperature with anti-PlxnA1 antibody (gift from Y. Yoshida), anti-Robo3 antibody (1/100, R and D, AF3076); anti-L1CAM antibody (1:100, A439 Abcam 123990), anti-NgCAM antibody (1:50, 8D9, DSHB), anti-BEN (1:50, BEN, DSHB) or an anti-PC2 antibody (1:100, 3533, Abcam) in 1% BSA diluted in PBS. Alexa 488, Alexa 555 (1/500, Invitrogen) and Fluoroprobe 546 (1/400) were used as secondary antibodies.

Immunocytochemistry was carried out as described previously^{56 (link)}. Cells were fixed with 4% paraformaldehyde/PBS for 15 min, permeabilized, and blocked with PBS containing 3% bovine serum albumin (A2153, Sigma-Aldrich) with 0.1% Triton X-100 (T9284, Sigma-Aldrich). Subsequently, cells were labeled with the primary ab, anti-MAP1LC3B (1/500) or anti-pH2AX (1/250). For negative controls, the primary ab was replaced with normal rabbit IgG or normal mouse IgG. After washing out the primary ab, cells were secondarily stained with Alexa Fluor 488 donkey anti-rabbit IgG (1/1000) or Alexa Fluor 488 donkey anti-mouse IgG (1/1000), followed with Hoechst33342 nuclear staining for 10 min. Cells were observed using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany).

For these assays, 14 mm round cover slides were placed in each well in a 24-well plate for hepatocytes to adhere to the wall. The cells were fixed with 4% paraformaldehyde (300 μL/well, P6148, Sigma-Aldrich) for 15 min, washed with PBS 3 times (5 min each), and perforated with 0.3% Triton X-100 (500 μL/well, T9284, Sigma-Aldrich) for 15 min to improve cell permeability. The cells were washed again with PBS three times, and then the sealant (3% BSA) was added to seal the samples in the incubator at 37 °C for 1 h. Then, primary antibodies p65 (1:100, AF0246, Beyotime), STIM1 (1:200, AF2614, Beyotime), Orai1 (1:200, 66223-1-Ig, ProteinTech), and Calnexin (1:100; AC019, Beyotime) were added, and the mixture was incubated overnight at 4 °C. After the cells were washed with PBS three times, the fluorescent secondary antibodies (1:500, A0562, and A0568, Beyotime) were added and incubated at 37 °C for 1 h without light. After three washes with PBS, the nuclei were stained with a DAPI solution (D8417; Sigma-Aldrich), and a round glass cover was fixed on the glass slide after washing for three times in the dark. The cells were observed by an LSM 710 confocal laser microscope system (Zeiss, Oberkochen, Germany).