Journal: Journal of Neurochemistry

Article Title: GABA B Receptor Modulation of Membrane Excitability in Human Pluripotent Stem Cell‐Derived Sensory Neurons by Baclofen and α‐Conotoxin Vc1.1

doi: 10.1111/jnc.70004

Figure Lengend Snippet: Generation of H9 NGN2 human pluripotent stem cell (hPSC)‐derived induced sensory neurons (iSNs). (A) Schematic protocol for the differentiation of iSNs. Briefly, hPSC are differentiated to neural crest cells (NCCs, days 1–6), then enriched for the cell surface marker CD271 via magnetic cell sorting. CD271 + NCCs are plated for sensory neuron differentiation (days 6–27), with induction of NGN2 expression via application of doxycycline for 96 h (days 6–10). (A) (I–IV) Representative micrographs of H9 NGN2 through differentiation to iSNs; (I) hPSC colonies, scale bar = 500 μm; (II) NCC after 6 days differentiation, scale bar = 500 μm; (III) 96 h NGN2‐induced neurite outgrowth, scale bar = 200 μm; (IV) iSNs after 3 weeks differentiation and maturation, scale bar = 50 μm. (B–F). Immunocytochemistry staining for molecular markers through hPSC differentiation to iSNs. (B) H9 NGN2 hPSC stained for pluripotency transcription factors SOX2 (green) and OCT4 (red). (C) H9 NGN2 ‐derived NCC stained for cell surface receptor CD271 (green) and transcription factor SOX10 (red). (D) iSNs stained for BRN3A (green) and ISL1 (red) transcription factors. (E) iSNs stained for neural marker TUJ1 (green) and peripheral neuronal marker PRPH (red). (F) iSNs stained for neurogenesis transcription factors NGN1 (green) and NGN2 (red).

Article Snippet: Antibodies used for immunocytochemistry were those against SOX2 (AF2018; R&D Systems), OCT4 (sc‐5279 Santa Cruz Biotechnology, Dallas, TX, USA), CD271 (M‐1818‐100; Biosensis, Thebarton, SA, Australia), SOX10 (AF2684; R&D Systems, Minneapolis, MN, USA), BRN3A (MAB1585; Millipore, St. Louis, MO, USA), ISLET1 (ab20670; Abcam, Cambridge, UK), TUJ1 (MAB1637, Millipore), TUJ1 (ab18207, Abcam), Peripherin (ab4666, Abcam), NGN1 (MA5‐24900, Invitrogen), NGN2 (PA5‐78556, Invitrogen), GABBR1 (AP23115PU‐N; OriGene, Rockville, MD, USA), GABBR2 (ab75838, Abcam), Ca V 2.2 (KP10001, CALBIOCHEM), GIRK1 (APC‐005; Alomone Labs, Jerusalem, Israel), Na V 1.7 (ab65167, Abcam), Na V 1.8 (ab66743, Abcam), S100β (ab52642, Abcam), MAP2 (M4403, Sigma‐Aldrich), and PSD95 (#51–6900, Invitrogen).

Techniques: Derivative Assay, Marker, FACS, Expressing, Immunocytochemistry, Staining, Cell Surface Receptor Assay

Journal: Cell Death & Disease

Article Title: HSCARG downregulates NF- κ B signaling by interacting with USP7 and inhibiting NEMO ubiquitination

doi: 10.1038/cddis.2014.197

Figure Lengend Snippet: HSCARG inhibits ubiquitination of NEMO. ( a ) HSCARG was coimmunoprecipitated with NEMO. HEK 293T cells were transfected with the indicated plasmids. At 48 h after transfection, cells were lysed and immunoprecipitated with anti-Flag antibody and then analyzed by immonoblotting with the indicated antibodies (left). The endogenous interaction between HSCARG and NEMO was examined by co-IP using anti-HSCARG antibody (middle). The effect of TNF α treatment on the HSCARG–NEMO interaction was assessed by co-IP as well (right). ( b – d ) HSCARG suppresses endogenous polyubiquitination of NEMO. HEK 293T cells were transfected with the indicated plasmids ( b and c ). Endogenous ubiquitin-conjugated NEMO was enriched and analyzed by His-ubiquitin pull-down assays. Polyubiquitination of NEMO was also examined in HSCARG −/− cells ( d )

Article Snippet: The shRNA plasmids were constructed by Shanghai Genechem Corporation (Shanghai, China). siRNA of NEMO (sc-29363) and IKK β (sc-35644) were from Santa Cruz (Dallas, TX, USA).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: HSCARG downregulates NF- κ B signaling by interacting with USP7 and inhibiting NEMO ubiquitination

doi: 10.1038/cddis.2014.197

Figure Lengend Snippet: HSCARG interacts with USP7, and inhibition of NEMO ubiquitination by HSCARG relies on the deubiquitination activity of USP7. ( a ) HSCARG interacts with USP7. Co-IP assays were performed to examine the interaction between HSCARG and USP7, CYLD or A20. HEK 293T cells were transfected with the indicated expression plasmids, and then whole-cell lysates were immunoprecipitated with anti-HA or anti-Flag antibodies, followed by immunoblotting with anti-Flag or anti-HA antibodies. WCL: 5% of whole-cell lysates. ( b ) Interaction between endogenous HSCARG and USP7 after TNF α treatment. HEK293T cells were treated with or without TNF α (5 ng/ml) for the indicated time, and then the endogenous interaction between HSCARG and USP7 was examined by co-IP analysis using anti-USP7 antibody. ( c ) NEMO is essential for the interaction of HSCARG with USP7. HEK 293T cells were transfected with control siRNA and NEMO siRNA (30 nM) for 72 h, and then treated with or without TNF α (5 ng/ml) for 20 min. The HSCARG–USP7 interaction was examined by co-IP using anti-USP7 antibody. ( d ) Inhibition of NEMO polyubiquitination by HSCARG is attenuated when USP7 is knocked down. HEK 293T cells were transfected with the indicated plasmids, and ubiquitin-conjugated NEMO was analyzed using His-ubiquitin pull-down assay

Article Snippet: The shRNA plasmids were constructed by Shanghai Genechem Corporation (Shanghai, China). siRNA of NEMO (sc-29363) and IKK β (sc-35644) were from Santa Cruz (Dallas, TX, USA).

Techniques: Inhibition, Ubiquitin Proteomics, Activity Assay, Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Control, Pull Down Assay

Journal: Cell Death & Disease

Article Title: HSCARG downregulates NF- κ B signaling by interacting with USP7 and inhibiting NEMO ubiquitination

doi: 10.1038/cddis.2014.197

Figure Lengend Snippet: USP7 interacts with NEMO and inhibits NEMO polyubiquitination, and HSCARG is important for the deubiquitination activity of USP7. ( a ) Co-IP assay was performed to investigate the interaction between USP7 and NEMO. HEK 293T cells were transfected with the indicated plasmids. At 48 h after transfection, cells were harvested and lysed, and the whole supernatant was immunoprecipitated with relevant antibodies and then analyzed by immunoblotting. ( b ) The interaction among USP7, HSCARG and NEMO was confirmed by co-IP assay. HEK 293T cells transfected with the indicated plasmids were lysed 48 h after transfection, and proteins were immunoprecipitated with the indicated antibody followed by immunoblotting (left). The interaction of NEMO with USP7 was examined in HSCARG −/− cells (middle). The effect of TNF α treatment on endogenous interaction among HSCARG, USP7 and NEMO was assessed by co-IP (right). ( c ) USP7 deubiquitinates NEMO. The wild-type and HSCARG −/− HEK 293T cells were transfected with the indicated plasmids, and His-ubiquitin pull-down assay was performed to assess the level of NEMO ubiquitination. ( d ) HSCARG regulates deubiquitination activity of USP7. Co-IP assay was performed in both HSCARG wild-type and HSCARG −/− HCT116 cells to examine the effect of USP7 on the level of ubiquitinated NEMO

Article Snippet: The shRNA plasmids were constructed by Shanghai Genechem Corporation (Shanghai, China). siRNA of NEMO (sc-29363) and IKK β (sc-35644) were from Santa Cruz (Dallas, TX, USA).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Pull Down Assay

Journal: Biochemistry

Article Title: A Central Region of NEMO is Required for IKKβ-Induced Conformational Change and for Signal Propagation

doi: 10.1021/acs.biochem.8b01316

Figure Lengend Snippet: The 9SG mutation abolishes the ability of NEMO to function in signal-induced activation of IκB kinase. (A) The indicated FLAG-tagged NEMO constructs were transfected into 293T cells, and extracts were immunoprecipitated (IP) with anti-FLAG beads. Immunoprecipitates were then analyzed by anti-FLAG (bottom) and anti-IKKβ (top) Western blotting. In the Input lanes, 4% of the extract used in the immunoprecipitations was analyzed by Western blotting. (B) Whole cell extracts from wild-type and NEMO-knockout 293T cells were analyzed by anti-NEMO Western blotting (top) or Ponceau staining for total protein (bottom). (C) 293T NEMO knockout cells were transfected with plasmids for the expression of 7XAla- or 9SG-NEMO. Cells that were either untreated (−) or treated with TNFα (+) were analyzed by Western blotting for phospho-IκBα or NEMO or by Ponceau staining. (D) Mouse NEMO knockout fibroblasts were stably transduced with retroviral vectors for the indicated NEMO proteins. Stable cell lines were then untreated (−) or treated (+) with the indicated compounds. Extracts were analyzed by Western blotting for the indicated proteins.

Article Snippet: In all cases, samples containing approximately equal amounts of protein were separated on SDS-polyacrylamide gels, proteins were transferred to nitrocellulose membranes, and filters were incubated overnight at 4 °C with NEMO antiserum (cat. #2685, Cell Signaling Technology; 1:1000 dilution) or with phospho-IκBα antiserum (cat. #9246, Cell Signaling Technology; 1:1000 dilution).

Techniques: Mutagenesis, Activation Assay, Construct, Transfection, Immunoprecipitation, Western Blot, Knock-Out, Staining, Expressing, Stable Transfection, Transduction