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. 2021 Nov 20;55:101400. doi: 10.1016/j.molmet.2021.101400

Figure 7.

Figure 7

FSTL1 exerted biological effects by inhibiting the integrin β1-mediated activation of the focal adhesion kinase pathway. (A, B) Heatmap of selected differential genes enrichment based on the RNA sequencing of the 3T3L1-NC and 3T3L1-shFstl1 groups after inducing for differentiation. (C) Gene Ontology analysis of differential genes in RNA-seq. (D) Western blot analysis of ITGB1 protein levels in the 3T3L1-NC and 3T3L1-shFstl1 groups after inducing for differentiation. GAPDH was used for loading control, ITGB1 band intensity was normalized relative to the GAPDH band. Student's t-test, ∗∗∗P < 0.005. (E, F) The culturing dishes were coated or not coated with Collagen Type I, and the protein levels of p-FAK (s397, s576/577, s925), FAK, p-ERK1/2 and T-ERK1/2 in 3T3L1 cells with Fstl1 knockdown (overexpression in F) and control group were analyzed by western blot. GAPDH was used for the loading control. (G) 293T cells were transfected with plasmids expressing Flag-tagged Fstl1 and HA-tagged Itgb1. Total cell lysate were subjected to IP with Flag or HA antibodies and immunoblotted for FSTL1 and ITGB1. Unspecific IgG antibodies were used as a control. (H) FSTL1 and ITGB1 protein complex was pulled down using glutathione-Sepharose beads and then subjected to western blot analyses with anti-FSTL1 antibody to confirm the presence of FSTL1 (upper). The presence of ITGB1 was detected with anti-ITGB1 antibody (lower). (I) 293T cells were transfected with HA-tagged Itgb1. The culturing dishes were coated or not coated with collagen Type I. ITGB1-HA was immunoprecipitated with HA-agarose and incubated with 100 ng FSTL1 protein. COL1 and ITGB1 were detected with anti-COL1 and anti-HA antibodies, respectively.