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. 2022 Jul 18;13(7):623. doi: 10.1038/s41419-022-05053-8

Fig. 5. CYB5A inhibits the STAT3 signaling pathway by inhibiting STOML2.

Fig. 5

A The whole-cell lysates obtained from HCC cell lines were co-immunoprecipitated by Flag antibody and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). And the Coomassie-stained SDS–PAGE analysis of washed beads. B The results were obtained and the top five proteins in PSM were selected for WB verification. C The whole-cell lysates obtained from untreated Huh-7 cell lines and Hep-G2 cell lines were co-immunoprecipitated by CYB5A antibody. D IF demonstrating the colocalization of CYB5A and STOML2 HCC cells, also shown in Supplementary fig. 5–1A. E HCCLM3-Con and HCCLM3-CYB5A cell lines transfected with HA-Ub were treated with Baf A1 and MG-132. The cell extracts were analyzed by co-IP and western blot. F Duolink PLA demonstrating the close proximity of CYB5A and STOML2 in HCC cells. G The expression changes of IL-6 and CYB5A after STOML2 overexpression were demonstrated by western blot assay (left panel). And The change of CYB5A expression in Huh-7 and HCCLM3 cells treated with IL-6 (20 ng/mL, 24 h) was demonstrated by WB assay (right panel). H STOML2 levels in lung xenograft tumors are shown by IHC. In our rescue test (in vivo, in vitro and at protein expression levels), we used lentivirus to regulate STOML2 expression to confirm that CYB5A regulates STOML2 (Supplementary figs. 5–1B, 5–2, 5–3, 5–4).