Fig. 5

Downregulation of pro-SFTPB expression upregulates PGK1 expression through ADRM1. a Silver staining showing the proteins that interact with pro-SFTPB in H1299 cells. Proteins that were bound to pro-SFTPB were pulled down by Co-IP using a pro-SFTPB antibody, separated by electrophoresis, and silver stained. The red arrow indicates the ADRM1 protein. b Co-IP analysis showing that pro-SFTPB interacts with ADRM1 in NSCLC cells. Flag-tagged pro-SFTPB was overexpressed in H1650 cells. Seventy-two hours after transfection, cell lysates were prepared, and co-IP was performed using antibodies against Flag or ADRM1. To detect the interaction between endogenous pro-SFTPB and ADRM1 proteins, the protein was extracted from H1299 cells and co-IP was performed using antibodies against pro-SFTPB or ADRM1. c Colocalization of pro-SFTPB and ADRM1 in NSCLC cells was demonstrated by immunofluorescence (IF) analysis. d Colocalization of PGK1 and ADRM1 in NSCLC cells was demonstrated by IF analysis. e Silencing of ADRM1 or UCH37 decreased PGK1 protein levels in NSCLC cells. The cells were subjected to Western blot analysis 72 h after transfection with the indicated siRNAs. f Downregulation of ADRM1 or UCH37 increases PGK1 ubiquitination in NSCLC cells. The cells were subjected to analysis 72 h after transfection with the indicated siRNAs. Co-IP was performed using an antibody against PGK1, and ubiquitinated PGK1 was detected by Western blotting. g Silencing of ADRM1 or UCH37 suppresses the pro-SFTPB-induced upregulation of PGK1 protein levels. Cells were subjected to Western blot analysis 72 h after transfection with the indicated siRNAs. h Silencing of ADRM1 or UCH37 suppresses the downregulation of pro-SFTPB-induced inhibition of PGK1 ubiquitination. The cells were subjected to analysis 72 h after transfection with the indicated siRNAs. Co-IP was performed using an antibody against PGK1, and ubiquitinated PGK1 was detected by Western blotting. sh pro-SFTPB, shRNA of pro-SFTPB; siADRM1, siRNA of ADRM1; siUCH37, siRNA of UCH37