Journal: The Journal of Neuroscience

Article Title: Assembly of GABA A Receptors Composed of α1 and β2 Subunits in Both Cultured Neurons and Fibroblasts

doi: 10.1523/JNEUROSCI.17-17-06587.1997

Figure Lengend Snippet: Differential sedimentation of the α1(9E10) subunit dependent on coexpression with the β2(9E10) subunit. Cells infected with the α1(9E10) (A) or co-infected with both the α1(9E10) and β2(9E10)(B) subunit viruses were lysed and subjected to sucrose density gradient fractionation 16 hr after infection. Gradient fractions were separated by SDS-PAGE, and the α1(9E10) subunit was detected via Western blotting using 9E10 antibody (A) or a rabbit polyclonal antibody against the α1 subunit (B). Sedimentation coefficients for the α1(9E10)subunit were determined with reference to proteins with known sedimentation: BSA (4.3 S), aldolase (7.4 S), and catalase (11.2 S).

Article Snippet: Receptor subunits were subjected to sucrose density gradient fractionation using 5 and 20% linear sucrose density gradients in lysis buffer ( Millar et al., 1995 ).

Techniques: Sedimentation, Infection, Fractionation, SDS Page, Western Blot

Journal: The Journal of Neuroscience

Article Title: Assembly of GABA A Receptors Composed of α1 and β2 Subunits in Both Cultured Neurons and Fibroblasts

doi: 10.1523/JNEUROSCI.17-17-06587.1997

Figure Lengend Snippet: Pulse–chase analysis of GABAAreceptor assembly. Cells co-infected with both subunit viruses were labeled with 100 μCi/ml [35S]methionine 2 hr after infection for 1 hr and chased for 0 (A), 6 (B), or 20 (C) hr excess cold methionine. The cells were then lysed and subjected to sucrose density gradient fractionation. Gradient fractions were then immunoprecipitated with 9E10 antibody, and the α1(9E10) and β2(9E10)subunits were resolved by SDS-PAGE. Sedimentation coefficients for the α1(9E10) and β2(9E10)subunits were determined as described in Figure ​Figure44.

Article Snippet: Receptor subunits were subjected to sucrose density gradient fractionation using 5 and 20% linear sucrose density gradients in lysis buffer ( Millar et al., 1995 ).

Techniques: Pulse Chase, Infection, Labeling, Fractionation, Immunoprecipitation, SDS Page, Sedimentation

Journal: The Journal of Neuroscience

Article Title: Assembly of GABA A Receptors Composed of α1 and β2 Subunits in Both Cultured Neurons and Fibroblasts

doi: 10.1523/JNEUROSCI.17-17-06587.1997

Figure Lengend Snippet: Subunit ratio of GABAA receptors composed of α1(9E10) and β2(9E10) subunits. A, BHK cells expressing both receptor subunits were pulse-labeled with [35S]methionine (200 μCi/ml) for 2 hr; 3 hr after infection, the cells were then chased for 12 hr with excess cold methionine. Cell lysates were then subjected to sucrose density gradient fractionation, and the fractions corresponding to the 9 S peak were pooled and immunoprecipitated with 9E10 antibody. Receptor subunits were then resolved by SDS-PAGE and quantified using a Bio-Rad phosphorimager. After correction for methionine content (α1 = 9; β2 = 15) a ratio of 0.9 was found for the α1(9E10):β2(9E10) subunits in the experiment shown. B, Labeled cells were exposed to 9E10 antibody at 4°C for 30 min. The cell surface receptor population was then isolated via immunoprecipitation with protein G in the presence of excess 9E10 peptide and resolved by SDS-PAGE, and subunit levels were quantified. A ratio of 1.1 was found for the α1(9E10):β2(9E10) subunits in the experiment shown.

Article Snippet: Receptor subunits were subjected to sucrose density gradient fractionation using 5 and 20% linear sucrose density gradients in lysis buffer ( Millar et al., 1995 ).

Techniques: Expressing, Labeling, Infection, Fractionation, Immunoprecipitation, SDS Page, Cell Surface Receptor Assay, Isolation

Journal: Cancer discovery

Article Title: Pharmacological suppression of B7-H4 glycosylation restores antitumor immunity in immune-cold breast cancers

doi: 10.1158/2159-8290.CD-20-0402

Figure Lengend Snippet: (A) SKBR3 cells were treated with 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. Membrane CALR, HSP70 and HSP90 were measured by flow cytometry. (B) MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells were established and treated with 5 μM doxorubicin for 24 h. Immunofluorescence staining of the immunogenic cell death markers CALR on the cell surface was performed. Mean fluorescence index of CALR was quantified by ImageJ. Representative images are shown. (C-D) SKBR3, MDA-MB-468, MDA-MB-468-vector, MDA-MB-468-B7-H4 knockout cells were treated with 1 or 10 μM doxorubicin and/or 10 μM NGI-1 for 24 h. p-eIF2a and actin were examined by immunoblotting. Scale bar, 100 μm. (E) Representative paired immunohistochemistry staining of B7-H4 and phospho eIF2α (Ser51) in tissue array BC081120. Statistical analysis of immunohistochemical staining indicates B7-H4 expression is negatively correlated with p-eIF2α expression in breast cancer (r = −0.249, p =8.71x10−3). (F) MDA-MB-468-Flag-hB7-H4 were treated in the presence or absence of doxorubicin (10 μM) and/or NGI-1 (10 μM). Then Flag-hB7-H4 was immunoprecipitated followed by immunoblot. The indicated proteins were examined. (G) Schematic diagram of the procedure of OptiPrep density gradient assay with 24 collected fractions from low to high density is shown. MDA-MB-468-vector and MDA-MB-468-hB7-H4 knockout cells were treated with 10 μM doxorubicin for 24 hr followed by OptiPrep density gradient assay. HSP90, CALR, eIF2α and p-eIF2α in fraction 1 to 13 were examined by immunoblotting. (H) eIF2a was immunoprecipitated in fraction 13 in both MDA-MB-468-vector and MDA-MB-468-B7-H4 knockout cells followed by immunoblotting. PERK, eIF2α and p-eIF2α were examined.

Article Snippet: OptiPrep density gradient protein fractionation assay To prepare the iodixanol gradient, a 50% (w/v) and 5% (w/v) solution of iodixanol were made by diluting the stock solution (60% (w/v) aqueous iodixanol from StemCell Technologies with 0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl pH7.4.

Techniques: Flow Cytometry, Plasmid Preparation, Knock-Out, Immunofluorescence, Staining, Fluorescence, Western Blot, Immunohistochemistry, Immunohistochemical staining, Expressing, Immunoprecipitation