Journal: The EMBO Journal

Article Title: ER-phagy restrains inflammatory responses through its receptor UBAC2

doi: 10.1038/s44318-024-00232-z

Figure Lengend Snippet: ( A ) Lysates of THP-1 Mφs (left) or HT-29 cells (right) treated with TG (1 μM) for 1 h in the presence of Baf A1 (0.2 μM) were subjected to immunoprecipitation and immunoblot analysis. ( B ) Lysates of THP-1 Mφs (left) or HT-29 cells (right) cultured in EBSS for 3 h together with Baf A1 (0.2 μM) treatment were subjected to immunoprecipitation and immunoblot analysis. ( C ) Lysates of HeLa cells treated with EBSS for indicated time points in the presence of Baf A1 (0.2 μM) were subjected to immunoprecipitation and immunoblot analysis. ( D ) Immunoblot analysis of the knockdown efficiency of MARK2 by MARK2 -specific siRNAs in HeLa cells. ( E ) HeLa cells were transfected with scramble or MARK2 -specific siRNA and treated with EBSS for 3 h in the presence of Baf A1 (0.2 μM). The lysates were subjected to immunoprecipitation and immunoblot analysis. ( F ) WT or MARK2 KO THP-1 Mφs were cultured in EBSS for 3 h with the existence of Baf A1 (0.2 μM). The lysates were treated with 2 mM disuccinimidyl suberate (DSS) cross-linker or immunoprecipitated using phosphoserine/threonine/tyrosine polyclonal antibody, and detected by immunoblot. ( G , H ) WT or MARK2 KO HT-29 cells were treated with TG (1 μM) for 1 h ( G ) and cultured in EBSS for 3 h ( H ) in the presence of Baf A1 (0.2 μM). The lysates were treated with 2 mM disuccinimidyl suberate (DSS) cross-linker or immunoprecipitated using phosphoserine/threonine/tyrosine polyclonal antibody, and detected by immunoblot. ( I ) 293T cells were transfected with scramble or MARK2 -specific siRNA and then transfected with vector of WT or S223A UBAC2. After incubation the cells with EBSS for 3 h in the presence of Baf A1 (0.2 μM), the lysates were harvested for immunoprecipitation and immunoblot analysis. ( J ) The band intensities of RFP and RFP-GFP in the similar samples as Fig. from three independent experiments performed in duplicate were quantified and the ratios of RFP/RFP-GFP were shown. ( K ) HeLa cells stably expressing the ER-phagy reporter were transfected with scramble or MARK2 -specific siRNA alongside with WT or mutated UBAC2 plasmids. The cells were then cultured in the presence of doxycycline for 24 h to induce the reporter and the protein was harvested for immunoblot analysis. ( L ) The band intensities of RFP and RFP-GFP in the similar samples as ( K ) from three independent experiments performed in duplicate were quantified and the ratios of RFP/RFP-GFP were shown. ( M ) HeLa cells stably expressing the ER-phagy reporter were transfected with scramble or UBAC2 -specific siRNA and then treated with PEP005 with indicated dosages for 1 h. The cells were cultured in EBSS for 3 h and the lysates were harvested for immunoblot analysis. Data information: For ( A – I , K , M ), one representative experiment out of three was shown. In ( J , L ), data are presented as the mean ± SEM of three independent biological experiments. The statistical significance of the difference was analyzed by unpaired two-tailed Student’s t test, and the P values were shown.

Article Snippet: Carfilzomib (cat. HY-10455), tunicamycin (cat. HY-A0098) and PEP005 (cat. HY-B0719) were purchased from MedChemExpress (MCE).

Techniques: Immunoprecipitation, Western Blot, Cell Culture, Knockdown, Transfection, Plasmid Preparation, Incubation, Stable Transfection, Expressing, Two Tailed Test