Fig. 6: PROCR-p62 interaction is diminished by mTOR-mediated p62 phosphorylation at S349.
From: PROCR diminishes the efficacy of radiation by impairing T-cell-mediated antitumour immunity
A Coimmunoprecipitation and immunoblot analysis of 293 T cells transfected with MYC-tagged PROCR together with FLAG-tagged p62, with or without radiation treatment, samples were collected at indicated timepoints after radiation. B Immunoblots showing phosphorylation of p62 at different sites at indicated timepoints after radiation treatment. C Flowcytometry analysis of PROCR in HONE1 cells transfected with PROCR and indicated wild-type p62 or its indicated mutants. D Coimmunoprecipitation and immunoblot analysis of 293 T cells transfected with indicated plasmids. E Immunofluorescence staining showing co-localization of PROCR and LC3B positive autophagosomes in HONE1 cells transfected with the indicated plasmids (n = 15 fields). Scale, 10μm. The boxplots indicate the median (centre), 25th, and 75th percentiles (box boundaries), and minimum and maximum (the whiskers). F Immunoblots analysis of PROCR in HONE1 cells transfected with p62-WT or p62-S349A mutant plasmids, treated with CHX for the indicated times. G RT-qPCR analysis of IL6 mRNA level of HONE1 cells transfected with wild-type p62 or its S349A mutant. H Immunoblots showing total and S349 phosphorylated p62 of HONE1 cells treated with DMSO, DMSO/SAR405(VPS34 inhibitor)/(5Z)−7-Oxozeanol (Tak1 inhibitor)/CKI7(CK1 inhibitor)/rapamycin (mTORC1 inhibitor) with radiation. I Co-immunoprecipitation and immunoblot analysis of 293 T cells transfected with MYC-tagged PROCR and FLAG-tagged p62, treated with radiation, with or without rapamycin (20 nM). J Immunoblots analysis of PROCR in HONE1 cells, treated with CHX for the indicated times, treated with radiation, with or without rapamycin (20 nM). K–M HONE1PROCR-Tet-On cells were inoculated into huHSC-NCG mice (1 × 106 HONE1 cells/mouse, 5 mice per group). 15 days after inoculation, mice were treated with irradiation (IR), irradiation + rapamycin (IR + Rapa), or irradiation + rapamycin + doxycycline (IR + Rapa + Dox). Tumour growth curves were monitored, and tumour volumes and weights were compared at the indicated time points. n = 3 independent experiments (A–J). One of three experiments is shown (A, B, D, F, H, I, J). Data are shown as mean ± SD (C, F, G, J) or mean ± SEM (L, M). P values were calculated using one-way ANOVA (C, E, G, M) or two-way ANOVA (F, J, L). Source data are provided as a Source Data file.
