Journal: Nature Communications

Article Title: Endothelial cell-derived stem cell factor promotes lipid accumulation through c-Kit-mediated increase of lipogenic enzymes in brown adipocytes

doi: 10.1038/s41467-023-38433-5

Figure Lengend Snippet: a , b Representative immunoblotting and comparisons of c-Kit, pAKT1 (Ser473), AKT1, pAKT2 (Ser474), AKT2, pERK1/2 (Thr202/Tyr204), ERK1/2, ACL, ACC, FASN, and SCD1 before and 10 min (10 m), 6 h, and 12 h after either bovine serum albumin (BSA, 100 ng/ml) or SCF (100 ng/ml) treatment in Control (transfected with empty lentiviral plasmid) or c-Kit over-expressed (transfected with c-Kit cDNA inserted lentiviral plasmid) cultured BAs. The cultured BAs were pre-incubated in high glucose DMEM containing 1% FBS for 12 h before and after the treatment. The same amount of protein loading in each lane is verified by immunoblotting of tubulin. Dots and bars indicate mean ± SD from n = 3/group from two independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001 versus BSA by two-tailed t-test. Protein sizes are indicated as kilodalton (kDa). c , d Representative immunoblotting and comparison of effect of indicated signaling inhibitor on the SCF-induced increased protein level of SCD1 in the c-Kit over-expressed and differentiated cultured BAs. The cultured BAs were pre-incubated in high glucose DMEM containing 1% FBS for 12 h before and after the treatment. For the inhibitor experiments, either Dynasore (20 µM), SU5402 (1 µM), wortmanin (30 nM), AKT1/2 inhibitor (25 nM), PD 0325901 (1 µM), ruxolitinib (10 µm) or Y27632 (10 mM) was treated to cultured cells for 1 hr, and then SCF (100 ng/ml) or VEGF (100 ng/ml) was added and incubated for 12 h. Each dot indicates a value from one experiment and n = 3/group from two independent experiments. Vertical bars indicate mean ± SD. * P < 0.05 versus SCF by one-way ANOVA test followed by Tukey’s post-hoc test. Protein sizes are indicated as kilodalton (kDa).

Article Snippet: After electrophoresis, proteins were transferred to a PVDF membrane, incubated with 2% bovine serum albumin (BSA) blocking buffer for 1 h at RT, and blotted with the following antibodies overnight: rabbit anti-c-Kit (#3074); rabbit anti-ACC (#3676); rabbit anti-FASN (#3180); rabbit anti-SCD1 (#2794); rabbit anti-GAPDH (#5174) (CST); rabbit anti-p-AKT1 (ser473) (#9018); rabbit-anti-AKT1 (#2938); rabbit-anti-p-AKT2 (Ser474) (#8599); rabbi-anti-AKT2 (#3063); rabbi-anti-p-p44/42 MAPK (Thr202/Tyr204) (#4377); rabbi-anti-p44/42 MAPK (#4695); rabbit anti-PGC1-α (#ab191838); rabbit anti-tyrosine hydroxylase (#ab137869); rabbit anti-ACL (#ab40793); rabbit anti-UCP1 (#ab23841) (Abcam) (4 h at RT or overnight); rat anti-α-tubulin (#sc-69970, Santacruz).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Cell Culture, Incubation, Two Tailed Test, Comparison