Phospho-STAT6 (Tyr641) Antibody - #AF3301

Figure 5 PARP14 deficiency activates the STAT1 pathway but blocks the STAT6 pathway in mice 7 days post-SCI. (A) Representative images and quantitative analysis showing p-STAT1 (Try701)+/Iba1+ immunofluorescence staining. Lv-shPARP14 injection further promoted SCI-induced STAT1 pathway activation. White arrows indicate p-STAT1 (Try701)+ (green, FITC-labeled)/Iba1+ (red, Cy3-labeled, microglia marker) cells. (B) Representative images and quantitative analysis showing p-STAT6 (Tyr641)+/Iba1+ immunofluorescence staining. Lv-shPARP14 injection inhibited SCI-induced STAT6 pathway activation. White arrows indicated p-STAT6 (Tyr641)+ (green, FITC-labeled)/Iba1+ (red, Cy3-labeled) cells. Scale bars: 50 µm. (C) Relative protein levels of p-STAT1 (Try701), and p-STAT6 (Tyr641) in each group were detected by western blot analysis. p-STAT1 (Try701) expression was increased by Lv-shPARP14 injection, but p-STAT6 (Tyr641) expression was decreased by PARP14 silencing. Values are shown as mean ± SD (n = 6). **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). Images were taken from the gray matter ventral horn at the injury site. Spinal cord tissues from the injury site were used for western blot detection. DAPI: 4′,6-Diamidino-2-phenylindole; Iba1: ionized calcium-binding adaptor molecule 1; PARP14: poly(ADP-ribose)polymerase, member 14; SCI: spinal cord injury; STAT1: signal transducer and activator of transcription.

Fig. 4. TRPM7 can mediate the macrophage polarization through transcription factors such as STAT1 and STAT6. (A and C) The protein levels of p-STAT1, STAT1, p-STAT6 and STAT6 were detected by Western blot assay. N ¼ 3; mean ± SD. (B and D) The ratios of p-STAT1 to STAT1 and the ratios of p-STAT6 to STAT6. N ¼ 3; mean ± SD. Control, untreated RAW264.7 cells. 2-APB, raw264.7 cells with 2-APB for 24 h. M1, RAW264.7 cells was stimulated with LPS- IFN-g for 24 h. M1þ2-APB, RAW264.7 cells was pretreated with 2-APB for 2 h and stimulated with LPS-IFN-g for 24 h. M2, RAW264.7 cells was stimulated with IL-4 for 24 h. M2þ2-APB, RAW264.7 cells was pretreated with 2-APB for 2 h and with IL-4 for 24 h. NC, RAW264.7 cells transfected with scrambled sequence. siRNA3, RAW264.7 cells transfected with siRNA3. M1þsiRNA3, RAW264.7 cells transfected with siRNA3-TRPM7 and stimulated with LPS-IFN-g for 24 h. M2þsiRNA3, RAW264.7 cells transfected with TRPM7-siRNA3 and stimulated with IL-4 for 24 h**P < 0.01, ***P < 0.0001 versus control; *P < 0.05, **P < 0.01, versus M1; &P < 0.05 versus M2.

Figure 6. |Stat signaling is involved in Wnt3A and IL-17A-mediated macrophage polarization in RAW264.7 cells. A, Representative images of immunoblots of indicated proteins of Stat signaling cascade in cells treated with different conditions. Semi-quantitative analysis of the expression of proteins in (A) showing fold changes of protein abundance of p-STAT1 (B), p-STAT3 (C), BCL-XL (D), p21(E), SOCS3 (F), p-STAT6 (G), and STAT6 (H) in cells treated with Wnt3a-CM and/or XAV939 in the presence or absence of rhIL-17A determined by densitometry assay using ImageJ software Fiji.