Figure 1
The expression of phosphorylated SFKs at Y416, Src, Fyn or Lyn in the ARC area following the CFA injection into the hind paw of rats. Gels shown in (a–c) were loaded with lysates prepared from the hypothalamic ARC region of animals without any treatment (Naïve) or at days 1, 3, 7 and 14 after the CFA (a,c) or normal saline [NS, (b)] injection into the hind paw as indicated. Each group of blots was cropped from a same filter (For an example see Fig. S2), stripped and successively probed with antibodies as indicated on the left of blots. Values on the right side of blots indicate the molecular mass (Kd). The ratio of band intensities versus that of GAPDH was calculated and then normalized to the ratio in naïve animals (=1, open bar) for determining relative changes. Bar graphs show summary data (mean ± SEM) of the relative changes. *, **p < 0.05, p < 0.01, Dunnett’s post hoc test in one-way ANOVA in comparisons with those of naïve rats. #,##p < 0.05, p < 0.01, Bonferroni’s post hoc test in two-way ANOVA in comparison between rats which received CFA versus saline injections into the hind pawl. Gels (from left to right) shown in (d–f) were loaded with immunoprecipitates performed using non-selective IgG from ARC tissues of naïve rats in the same experimental sets, or using antibodies recognizing Src, Fyn or Lyn from ARC tissues of rats at day 3 after the injection of normal saline or CFA into the hind pawl. The group of blots shown in (d) or (e) was cropped from a same filter, and the group of blots shown in (f) was from the full length filter shown in Fig. S5. Each group of blots was stripped and successively probed with antibodies as indicated: the SFK-pY416 antibody for top blots in (d–f); the Src, Fyn and Lyn antibodies (which were used to precipitate Src, Fyn and Lyn, respectively) were used to reprobe the filters shown in middle blots in (d–f), respectively; the Fyn antibody was used to probe the filter shown in the bottom blot in (d) and the Src antibody to probe the filter shown in the bottom blots in (e,f). Bands detected in the immunoprecipitates of non-selective IgG indicate positions of heavy chains of the antibodies used. The original blots of those displayed in (d–f) are shown in Figs S3–S5, respectively. The ratios of the band intensities of SFK-pY416 versus that of Src, Fyn and Lyn immunoprecipitated were respectively calculated. Bar graphs show summary data. NS: rats which received the injection of normal saline into the hind paw. CFA: rats which received the CFA injection into the hind paw. #p = 0.038, t6 = 2.64, unpaired t-test in the comparison of rats receiving the injection of normal saline versus ACF into the hind paw. Values in brackets indicate the number of experimental repeats.
