anti ddb1  (Proteintech)

Journal: Science Advances

Article Title: Cellular stress triggers degradation and global redistribution of RNAPII

doi: 10.1126/sciadv.aec6649

Figure Lengend Snippet: ( A ) RNAPII Ser-2P specific IP-MS assay was performed in both control (PBS, 2 hours) and stressed (120 mM KCl, 2 hours) 293 T cells, the Cullin E3 complex related candidates were depicted by their enrichment intensity and enrichment foldchanges (the protein enrichment intensity from stressed cells was divided by that of the control). ( B ) and ( C ) RNAPII Ser-2P specific immunoprecipitation was performed in non-treated 293 T control and KCl (120 mM KCl, 2 hours) treated cells (2-hour MG-132 pre-incubation before KCl treatment was used in to prevent extensive RNAPII Ser-2P degradation). Protein levels of RNAPII Ser-2P, CUL1, FBXO11, DDB1 and CUL4B in the eluate were detected by their corresponding antibodies. ( D ) 293 T cells transfected with HA-FBXO11 coding plasmid for 12 hours, were then incubated with MG-132 for 2 hours, followed by 80 mM KCl. These KCl treated and control cells were stained by RNAPII Ser-2P and HA antibodies, and were visualized by iSTORM supper resolution microscopy (wide field similar with confocal capture). The statistic analysis of colocalization were shown as fluorescent intensity track and signal correlation.

Article Snippet: The following antibodies were used: RNAPII Ser-2P (Abcam, 1:2000), β-tubulin (Abcam, 1:2000), HA (Abcam, 1:2000), FLAG (Abcam, 1:2000), pATM (Abcam, 1:1000), Ubiquitin (Invitrogen, 1:1000), BCL2 (ABclonal, 1:1000), CASP3 (ABclonal, 1:1000), DDB1 (ABclonal, 1:1000), CUL4B (ABclonal, 1:1000), FBXO11 (ABclonal, 1:1000), CUL1 (ABclonal, 1:1000), TNFα (ABclonal, 1:1000), p38 (ABclonal, 1:1000), Phosphorylated-p38 (ABclonal, 1:1000), Phosphorylated-JNK (ABclonal, 1:1000), CUL4A (ABclonal, 1:1000), RNAPII Ser-5P (ABclonal, 1:1000), RNAPII (ABclonal, 1:1000), γH2AX (ABclonal, 1:1000), ATM (ABclonal, 1:1000), DNAPKcs (ABclonal, 1:1000), pDNAPKcs (ABclonal, 1:1000); Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP (Invitrogen, 1:3000), Goat anti-Rabbit IgG (H + L) Secondary Antibody HRP (Invitrogen, 1:3000).

Techniques: Protein-Protein interactions, Control, Protein Enrichment, Immunoprecipitation, Incubation, Transfection, Plasmid Preparation, Staining, Microscopy

Journal: Advanced Science

Article Title: Halofuginone is a Molecular Glue Degrader of Integrin β 4

doi: 10.1002/advs.202515970

Figure Lengend Snippet: HF promotes proteasomal degradation of integrin β 4 by conferring glue activity. (a) Western blot to determine the degradation rates of integrin β 4 by HF exposure (0.5 µM) after adding protein synthesis inhibitor CHX (300 µM) in KYSE30 and KYSE450 cell. (b) Western blot was performed to find the pathway of HF induced integrin β 4 degradation in KYSE30 and KYSE450 cell, wortmannin was use to inhibit autophagy. (c) Western blot to determine the ubiquitination of integrin β 4 upon HF administration in ESCC cells, briefly, KYSE30 and KYSE450 cell were exposed to different concentrations of HF (0–2 µM), then, total cell extracts were subjected to IP using an anti‐integrin β 4 antibody, ubiquitin was detected in the immunoprecipitates by western blot. (d, e) Cullin associated proteins and potential substrate receptors with the highest scores in mass spectrometry result. (f, g) Western blot was performed to test whether knockdown of these proteins would result in integrin β 4 abundance change within or without HF exposure. (h) IP‐western blot was performed to identify which substrate receptors could bind to DDB1. Briefly, the reconstituted plasmids containing a 3×flag tag on the N terminal of WDR18, WDR26 and WDR57, were transfected to HEK293T cell, then, total cell extracts were subjected to IP using Anti‐flag magnetic beads, flag tag and DDB1 was detected in the immunoprecipitates by western blot. (i) Western blot analysis of integrin β 4 abundance in the immunoprecipitates enriched by anti‐flag magnetic beads in HEK293T cell that transfected with Flag‐tagged WDR18. (j) Western blot analysis of ubiquitinzed integrin β 4 after knockdown of CRL4B WDR18 components. (k) Streptavidin‐pulldown and western blot were performed to determine the binding ability of HF to CRL4 WDR18 E3 ligase components. (l) Co‐IP and western blot analysis of the binding affinity between integrin β 4 and WDR18 upon HF and PEG‐HF administration in KYSE30 cell. (m) Construction of truncated ITGB4 plasmids and the mutant plasmid. (n) HEK293T cells were transfected with ITGB4‐C1, ITGB4‐C2 or ITGB4‐C3, and then the transfected cells were exposed to DMSO, biotin, HF and PEG‐HF, respectively, HA tag was detected by western blot. (o) HEK293T cells were transfected with ITGB4‐C2 or ITGB4‐C3 for 24 h, then the transfected cells were treated with PEG‐HF, total cell extracts were subjected to IP using an anti‐HA antibody, WDR18 and HA tag were detected in the immunoprecipitates by western blot. (p) IP‐western blot was performed to test whether the key sites mutation in ITGB4‐C2 would disrupt its binding to HF. Briefly, HEK293T cells were transferred with ITGB4‐C2 and ITGB4‐C2M, respectively. The transferred cells were treated with HF overnight. Total cell extracts were subjected to IP using an anti‐HA antibody, WDR18 and HA tag were detected in the immunoprecipitates by western blot.

Article Snippet: Antibodies against DDB1 (A3827), DCAF7 (A6787), WDR18 (A15875) were purchased from ABclonal.

Techniques: Activity Assay, Western Blot, Ubiquitin Proteomics, Mass Spectrometry, Knockdown, Transfection, Magnetic Beads, FLAG-tag, Binding Assay, Co-Immunoprecipitation Assay, Mutagenesis, Plasmid Preparation