Journal: International Journal of Biological Sciences

Article Title: Identification of Novel Focal Adhesion Kinase Substrates: Role for FAK in NFκB Signaling

doi: 10.7150/ijbs.10273

Figure Lengend Snippet: Microarray screen for FAK substrates. a. Left: Autoradiograph image of control and FAK protein microarray slides. Right: Control microarray containing 1, 5, 10 and 25 ng/dot of GST or GST-Paxillin. b. Spot-pair signals from control and FAK slides block #40 (upper panels), with identified proteins, microarray positions and relative signal intensities (average pixels/pair) below. A-F, proteins with increased phosphorylation by FAK relative to control; G, positive control protein, PKCeta; H-J , proteins with equal or decreased phosphorylation levels in the FAK slide compared to control.

Article Snippet: The following reagents/kits were used: ProtoArray Human Protein Microarray v5.0 Kinase Substrate Identification Complete Kit (Invitrogen), full-length active Src (SignalChem), GST-FAK (Invitrogen), baculovirus-encoded Pyk2 (gift of Vita Golubovskaya, Roswell Park Cancer Institute [RPCI]), purified vitronectin (Advanced BioMatrix), purified GST-PTPN5[a.a.17-565] (ProteinTech), FAK inhibitor PF-573,228 (Sigma), TNFα (Abcam), Recombinant Light (Enzo Life Sciences).

Techniques: Microarray, Autoradiography, Blocking Assay, Positive Control

Journal: eLife

Article Title: RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells

doi: 10.7554/eLife.63452

Figure Lengend Snippet: ( A, B ) Mouse Clec9A binds to the RNF41 C-terminal domain. ELISA plates were coated with GST-tagged RNF41-C-terminal domain (RNF41-C), RNF41-N-terminal domains (RNF41-RBC) and GST control. ( A ) Binding of FLAG-tagged mClec9A-ecto or the control FLAG-mClec12A-ecto (10 μg/ml) was detected with anti-FLAG HRP. Cumulative data of three experiments is shown, presented as mean ± SEM (unpaired t-test) ****p<0.0001. ( B ) Binding of FLAG-tagged mClec9A-ecto or the control FLAG-mClec12A-ecto (10 μg/ml to 0.04 μg/ml) was detected with anti-FLAG HRP. Cumulative data of three experiments is shown. ( C, D ) Mouse Clec9A binding to RNF41 is enhanced at low pH. ELISA plates were coated with GST-tagged RNF41-C, actin complexes or GST. Binding of biotinylated FLAG-tagged mClec9A-ecto or biotinylated control FLAG-mClec12A-ecto at different pH was detected with streptavidin-HRP. Data is presented as the absorbance 405–490 nm of (Clec binding to GST-RNF41 - Clec binding to GST). ( C ) Cumulative data of three experiments is shown, presented as mean ± SEM. mClec9A (2.5 μg/ml) binding to RNF41-C at pH 4, pH 5 and pH 6 was significantly greater than at pH 7.4 and at pH 8 (1-way ANOVA) *p<0.05. ( D ) Representative of three independent experiments. Figure 1—source data 1. Clec9A binding to RNF41.

Article Snippet: Commercial assay or kit , ProtoArray Human Protein MicroArray Version 4.1 PPI Kit for biotinylated proteins , Thermo Fisher , Cat# PAH05241011 , .

Techniques: Enzyme-linked Immunosorbent Assay, Control, Binding Assay

Journal: eLife

Article Title: RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells

doi: 10.7554/eLife.63452

Figure Lengend Snippet: ( A, B ) RNF41 mediates mClec9A ubiquitination in vivo. 293 F cells were co-transfected with mClec9A-FLAG or hCLEC9A-FLAG, Myc-tagged ubiquitin (Ub-Myc), and RNF41 or RNF41-ΔRING. At 22 hr, IP Clec9A complexes were analyzed for ubiquitination by WB using anti-Ubiquitin Ab, representative of ( A ) 10 experiments, ( B ) seven experiments, and anti-Ubiquitin K-48 Ab, representative of ( A ) two experiments. Densitometric analysis of ubiquitinated Clec9A relative to total Clec9A from IP of Clec9A complexes from ( A ) 10 experiments and ( B ) seven experiments is shown, as mean ± SEM (ANOVA) **p<0.01, ****p<0.0001. ( C ) RNF41 mediates Clec9A ubiquitination in vitro. mClec9A-FLAG was immunoprecipitated from transfected 293 F cells, then incubated with GST-RNF41 or GST-RNF41-C in the presence of E1, E2 (UbcH5a) and biotinylated ubiquitin. Ubiquitination of Clec9A complexes was analyzed by WB for ubiquitin (Streptavidin-HRP), total Clec9A (anti-FLAG-M2) and total RNF41. Representative of two independent experiments. ( D, E ) Proteomic analysis of RNF41 mediated ubiquitination of Clec9A complexes. 293 F cells were co-transfected with mClec9A-FLAG, Ub-Myc and RNF41 or RNF41-ΔRING. At 22 hr, IP Clec9A complexes (pH 6.0) were analyzed by LC-MS/MS. ( D ) Nine major Clec9A ubiquitination sites were identified, from at least four independent experiments, and the position of these sites is indicated. ( E ) Heat map showing quantification results of 6 and 3 ubiquitinated peptides derived from Clec9A and Ubiquitin, respectively, across three independent experiments as indicated by 1,2,3 above the heat map. The intensities of the ubiquitinated peptides are shown as log2. An X indicates that the ubiquitinated peptide was not confidently detected or quantified. Figure 4—source data 1. RNF41 mediates ubiquitination of mouse and human Clec9A.

Article Snippet: Commercial assay or kit , ProtoArray Human Protein MicroArray Version 4.1 PPI Kit for biotinylated proteins , Thermo Fisher , Cat# PAH05241011 , .

Techniques: Ubiquitin Proteomics, In Vivo, Transfection, In Vitro, Immunoprecipitation, Incubation, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: eLife

Article Title: RNF41 regulates the damage recognition receptor Clec9A and antigen cross-presentation in mouse dendritic cells

doi: 10.7554/eLife.63452

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , ProtoArray Human Protein MicroArray Version 4.1 PPI Kit for biotinylated proteins , Thermo Fisher , Cat# PAH05241011 , .

Techniques: Expressing, Transgenic Assay, Transfection, Construct, Recombinant, Synthesized, Confocal Microscopy, Ubiquitin Proteomics, Plasmid Preparation, Knockdown, shRNA, Bacteria, Sequencing, Positive Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Bicinchoninic Acid Protein Assay, Microarray, Labeling, In Situ, Software