Journal: bioRxiv

Article Title: ZFAND6 is a subunit of a TRAF2-cIAP E3 ubiquitin ligase complex essential for mitophagy

doi: 10.1101/2024.03.27.586763

Figure Lengend Snippet: A) Confocal microscopy with fluorescently conjugated antibodies to pUb using WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for 3 hr. in the presence of 100 μM leupeptin. Scale bars represent 5 μm. Data presented as mean ± SEM. ( B, C) Co-IP assays were performed with lysates from 293T cells transfected with the indicated plasmids. Immunoprecipitations and western blotting were performed with the indicated antibodies to examine ZFAND6-TRAF2 (B) and ZFAND6-cIAP1 (C) interactions. (D) Co-IP assay was performed with lysates from WT and Zfand6 −/− BMDMs treated with 5 μM oligomycin A and 1 μM antimycin A for the indicated times. IPs were performed with either isotype control antibody or TRAF2 antibody. Western blotting was performed with the indicated antibodies. Unpaired Student’s t-test. ****P<0.0001.

Article Snippet: ZFAND6-Flag-Myc was purchased from Origene. pEBB-HA cIAP1 was a gift from Colin Duckett (Addgene plasmid# 38232; http://n2t.net/addgene:38232 ; RRID:Addgene_38232). lentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid# 52691 ; http://n2t.net/addgene:52691 ; RRID:Addgene_52691). pUltra-Hot was a gift from Malcolm Moore (Addgene plasmid# 24130 ; http://n2t.net/addgene:24130 ; RRID:Addgene_24130). pUltra-Hot-ZFAND6 was generated by subcloning a ZFAND6 PCR product digested with Xba1 (New England Biolabs; R0145S) and BamH1-HF (New England Biolabs; R3136S) into pUltra-Hot.

Techniques: Confocal Microscopy, Co-Immunoprecipitation Assay, Transfection, Western Blot, Control

Journal: Human Molecular Genetics

Article Title: XIAP and cIAP1 amplifications induce Beclin 1-dependent autophagy through NFκB activation

doi: 10.1093/hmg/ddv052

Figure Lengend Snippet: cIAP1 overexpression induces autophagy. ( A ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were treated with DMSO or 400 n m bafilomycin A1 (Baf A1) during the last 4 h. Blots were probed with the indicated antibodies and the HA indicates the cIAP1 constructs. Densitometric measurements of LC3-II bands were normalized to the corresponding actin bands and are shown in the histogram on the right. ( B ) HeLa cells stably expressing mRFP-GFP-LC3 transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were fixed and subjected to automatic counting of LC3 vesicles. The histogram shows the percentage relative to C- of the number/cell of autophagosomes (mRFP+/GFP+) (AP), autolysosomes (mRFP+/GFP-) (AL) and both of them (total). ( C ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were subjected to western blotting. Densitometric measurements of p62 bands were normalized to the corresponding actin bands and are shown in the histogram on the right. ( D ) HeLa cells were co-transfected with the GFP-HttQ74 expression construct plus empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h. The cells were then fixed and the percentage of transfected cells with aggregates was calculated as shown in the histogram. At least 150 cells were counted per sample. The values shown in all the histograms represent the mean ± standard deviation from at least three independent experiments performed in triplicate samples/condition. The P -values were determined using Student's t -test. See also Supplementary Material, Figure S8 .

Article Snippet: Ashwell (Addgene plasmid 11559) , pEBB-HA-cIAP1 and pEBB-HA-cIAP1 H588A were provided by C.S.

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Construct, Stable Transfection, Western Blot, Standard Deviation

Journal: Human Molecular Genetics

Article Title: XIAP and cIAP1 amplifications induce Beclin 1-dependent autophagy through NFκB activation

doi: 10.1093/hmg/ddv052

Figure Lengend Snippet: cIAP1 induces Beclin 1 transcription via p65/NFκB activation. ( A ) HeLa cells were transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h. Densitometric measurements of Beclin 1 bands were normalized to the corresponding actin bands and are shown in the histograms on the right. ( B ) mRNA from HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h was analysed by qRT-PCR for Beclin 1-actin mRNA. The levels of Beclin 1 mRNA were normalized to Actin mRNA levels. ( C ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were subjected to western blotting to detect P-IκB and IκB levels. The blots shown are from the same set of experiments. Densitometric measurements of phospho-IκB (P-IκB) bands were normalized to the corresponding bands of actin and are shown in the histogram below. ( D ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were subjected to a ChiP assay. The amount of in vivo binding of endogenous p65 to Beclin 1 and actin (as a negative control) promoters was quantified by real-time PCR. Data are representative of three independent experiments. The values shown in all the histograms represent the mean ± standard deviation from at least three independent experiments performed in triplicate samples/condition. The P -values were determined using Student's t -test.

Article Snippet: Ashwell (Addgene plasmid 11559) , pEBB-HA-cIAP1 and pEBB-HA-cIAP1 H588A were provided by C.S.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Construct, Quantitative RT-PCR, Western Blot, In Vivo, Binding Assay, Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation