Journal: Nature Communications

Article Title: The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation

doi: 10.1038/s41467-019-09299-3

Figure Lengend Snippet: The NxNNWHW motif is required for Aha1p function in vivo. a Yeast expressing Hsc82p S25P as the sole source of Hsp90 in the cell exhibit temperature-sensitive growth. The deletion of AHA1 in yeast expressing Hsc82p S25P exacerbates the temperature-sensitive growth defect. b Overexpression of myc-tagged Aha1p, but not Aha1p Δ11 , rescues the growth of yeast expressing Hsc82p S25P . Yeast expressing Hsc82p S25P and harboring expression plasmids encoding the indicated co-chaperones were grown overnight at 30 °C in YPD supplemented with 300 mg/L Hygromycin and then diluted to 1 × 10 8 cells per milliliter. We prepared 10-fold serial dilutions and spotted 10 μL aliquots on YPD agar plates supplemented with Hygromycin 300 mg/L. Plates were incubated for 2 days at 30, 34, or 37 °C. c Western blot of total lysates and soluble protein extracted from the yeast strains shown in b were probed with anti-Hsp90, anti-actin, and anti-myc antibodies. d Overexpression of Aha1p, but not Aha1p Δ11 , enhances v-src activation in yeast expressing Hsc82p S25P . Yeast expressing Hsc82p S25P and harboring expression plasmids encoding the indicated co-chaperones and a galactose inducible v-src expression plasmid were grown overnight at 30 °C in SC-Ura containing 2% raffinose, supplemented with 300 mg/L hygromycin. Cells were diluted to an OD 600 of 0.5 and grown for an additional 6 h in SC-Ura containing either 2% glucose or galactose, supplemented with hygromycin (300 mg/L). Yeast strains were probed with anti-Hsp90, anti-myc, anti-v-src, anti-phosphotyrosine, and anti-actin antibodies. Representative results of three independent experiments are shown

Article Snippet: Myc-tagged proteins were detected with mouse anti-myc monoclonal antibody (1:100; 4A6 Millipore, catalog number 05-724) and Hsp82p was detected with anti-Hsp90 antibody (1:1000; Anti-Hsp90, Clone K41220A, Stressmarq Biosciences Inc., Victoria, BC, Canada; catalog number SMC-135), v-src was detected with anti-v-src antibody (1:200; clone 327 Sigma-Aldrich; catalog number MABS193), phosphotyrosine levels were detected with anti-phosphotyrosine antibody (1:1000; Stressmarq Biosciences Inc., Victoria, BC, Canada; catalog number SMC-157).

Techniques: In Vivo, Expressing, Over Expression, Incubation, Western Blot, Activation Assay, Plasmid Preparation

Journal: NPJ Breast Cancer

Article Title: The human intermediate prolactin receptor is a mammary proto-oncogene

doi: 10.1038/s41523-021-00243-7

Figure Lengend Snippet: MCF10AT transfectants were PRL (250 ng/mL) stimulated for 0, 15, and 30 min, and phospho-IB was utilized to assess the activation status of a , b Jak2/Stat5a and c , d Mek/Erk. Band intensities were quantified using densitometry. Significance was determined by comparing each condition to the MCF10AT-EV negative control for each respective time point. * p < 0.05, n = 3.

Article Snippet: Antibodies used for these studies were obtained from the following sources at the indicated titer: hPRLr ECD (35–9200, Invitrogen, 1:1000), pY-Stat5a (9359S, Cell Signaling, 1:1000), Stat5a (sc-1081, Santa Cruz Biotechnology, 1:1000), pY-Jak2 (3776S, Cell Signaling, 1:500), Jak2 (3230S, Cell Signaling, 1:500), p-p44/42 (9101S, Cell Signaling, 1:1000), p44/42 (9102S, Cell Signaling, 1:1000), pS-Mek (9121S, Cell Signaling, 1:1000), Mek (9122S, Cell Signaling, 1:1000), KRAS (14412S, Cell Signaling, 1:1000), hPRLrI (New England Peptide, 1:5000), pS349-hPRLr (Serge Y. Fuchs, M.D., Ph.D., University of Pennsylvania, 1:100), Vinculin (MCA465GA, Bio-Rad, 1:1000).

Techniques: Activation Assay, Negative Control

Journal: Molecular Medicine

Article Title: Vagal afferent fibers contribute to the anti-inflammatory reactions by vagus nerve stimulation in concanavalin A model of hepatitis in rats

doi: 10.1186/s10020-020-00247-2

Figure Lengend Snippet: Regulation on the production of α7 nAChRs and pY-STAT3 in the liver by the inhibition of afferent vagus nerve activity in ConA-injected animals. a Western blot analysis of liver tissue lysates from animal groups as indicated in the figure. A representative image from 4 independent experiments is shown (upper), and a quantification of pY-STAT band intensity relative to actin is plotted (lower). Western blotting for actin as sample loading control. b Immunofluorescence localization of pY-STAT3 and CD11b in the liver tissue. Perinuclear localization of pY-STAT3 was identified by Hoechst nuclear staining (blue). A percentage of the number of pY-STAT3-immunostained cells relative to CD11b-labeled cells was plotted (right). c , d Western blot analysis of α7 nAChRs ( c ) and pY-STAT3 proteins ( d , e ) for protein lysates of liver tissue. Upper images are the representatives from 4 independent experiments. AP/CN; a group injected with AP-5/CNQX into the DMV. Keta/Xyla; ketamine/xylazine. In a – e , one way ANOVA with Tukey post hoc is shown. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar in b = 50 μm. f A schematic showing the proposed afferent vagus nerve pathway activated by VNS that contributes to cholinergic anti-inflammation in the liver. Afferent and efferent neve activity induced by VNS are shown by red and blue arrows respectively. NG; nodose ganglion, NTS; nucleus tractus solitarius, DMV; dorsal motor nucleus of the vagus nerve, PGN; postganglionic neuron

Article Snippet: Immunoblotting was performed with primary antibodies against TNF-α (ab9739, Rabbit-polyclonal, 1:2,000, Abcam, Cambridge, UK, RRID:AB_308774), IL-1β (ab9722, Rabbit-polyclonal, 1:2,000; Abcam, RRID:AB_308765), IL-6 (ab9324, Mouse-monoclonal, 1:2000; Abcam, RRID:AB_307175), ChAT (ab181023, Rabbit-monoclonal, 1:2000, Abcam, RRID:AB_2687983), pY-STAT3 (#9145, Rabbit-polyclonal, 1:1000; Cell Signaling Technology, Danvers, MA, USA, RRID:AB_2491009), α7 nAChR (ANC-007, Rabbit-polyclonal, 1:200, Alomone Labs, Jerusalem, Israel), and β-actin (A1978, Mouse-monoclonal, 1:50,000; Sigma-Aldrich, RRID:AB_476692).

Techniques: Inhibition, Activity Assay, Injection, Western Blot, Control, Immunofluorescence, Staining, Labeling

Journal: The Application of Clinical Genetics

Article Title: Hemizygous IL2RG Variants Impair IL-2-Induced STAT5 Phosphorylation and Transcriptional Activity Causing X-Linked Severe Combined Immunodeficiency

doi: 10.2147/TACG.S525027

Figure Lengend Snippet: Reduced IL2RG expression on the cell surface affects IL-2-mediated signal transduction. ( A ) IL2RG mutant protein expression in HEK293 cells, as determined by Western blotting. ( B ) Co-IP analysis showed significantly reduced binding between JAK3 and the three IL2RG mutant proteins (p.R190P, p.L172P, and p.T364I), while the p.T73P mutant protein exhibited partial interaction with JAK3. ( C ) Flow cytometry analysis demonstrated that the surface expression of mutant IL2RG proteins was significantly reduced in HEK293 cells compared to that of wild-type IL2RG. ( D ) After stimulation with 1000 U/mL IL-2, STAT5 phosphorylation was substantially decreased in cells expressing mutant IL2RG proteins compared to wild-type IL2RG. ( E ) Luciferase reporter assays showed a significant reduction in STAT5 transcriptional activity in cells expressing the mutant IL2RG proteins compared to those expressing wild-type IL2RG. Cells in the blank control group were untransfected with any plasmid. Data are representative of three or four independent experiments, results are represented as mean ± SD, and statistical analyses were carried out via t test. (** p <0.01; *** p <0.001; **** p <0.0001).

Article Snippet: Membranes were incubated overnight at 4 °C with primary antibodies against Flag-tag, Myc-Tag (1:1000, Abways Technology, AB0001, Shanghai, China), Stat5 (D2O6Y) Rabbit mAb (1:1000, Cell Signaling Technology, 94205, Danvers, MA, USA), Phospho-Stat5 (Tyr694) (D47E7) XP Rabbit mAb (1:1000, Cell Signaling Technology, 4322, Danvers, MA, USA), and GAPDH (1:1000, Affinity Biosciences, T0004, Liyang, Jiangsu, China).

Techniques: Expressing, Transduction, Mutagenesis, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Flow Cytometry, Phospho-proteomics, Luciferase, Activity Assay, Control, Plasmid Preparation