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. Author manuscript; available in PMC: 2019 Jun 28.
Published in final edited form as: Cell. 2018 Jun 14;174(1):156–171.e16. doi: 10.1016/j.cell.2018.05.027

Figure 2. LRRC33 association with proTGF-β1 and TGF-β1 activation.

Figure 2.

(A and B) Lysates of 293T cells transfected with indicated constructs (A) or culture supernatants (B) were immunoprecipitated (IP) and subjected to reducing SDS 10% PAGE and blotted (WB) as indicated. (C) Disulfide linkage. 293T cells transfected with indicated constructs were subjected to IP, 7.5% non-reducing or 10% reducing SDS-PAGE, and WB as indicated. (D) LRRC33 outcompetes LTBP for proTGF-β1 293T transfectant lysates were IP, subjected to non-reducing SDS 7.5% PAGE, and WB as indicated. (E) LRRC33-proTGF-β1 complex in THP-1 cells. THP-1 cells were treated with or without PMA (80 nM, 24 h) and cell lysates were IP with 1/8.8 to LRRC33 or mouse IgG control, reducing and non-reducing SDS 7.5% PAGE, and WB as indicated. (F) Flow cytometry. THP-1 cells treated with or without PMA were stained with anti-LRRC33 (1/8.8), anti-prodomain (TW4–2F8), anti-integrin αV (17E6) or anti-integrin β6 (7.1G10) and subjected to FACS. Numbers in histograms show specific mean fluorescence intensity. (G) Blockade of active TGF-β1 release. THP-1 cells treated with or without PMA were incubated with antibody 1/8.8 to LRRC33, 17E6 to αV integrin, or MAB240 to TGF-β1 and cocultured with TMLC to measure TGF-β activation. Data represent mean ± SEM of quadruplicate samples.