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: CNS Neuroscience & Therapeutics

Article Title: ErbB2 pY ‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

doi: 10.1111/cns.14407

Figure Lengend Snippet: Western blot analysis of ErbB2 pY‐1248 , ErbB2, and ErbB1 in cellular PD model. (A, C) Representative images of western blot of ErbB2 pY‐1248 , ErbB2, and ErbB1 in extracted protein lysates from SH‐SY5Y cells treated with 6‐OHDA for 6 h. (B, D) Grayscale value analysis of the immunoblots. The band intensity of ErbB2 pY‐1248 and ErbB1 were normalized to ErbB2 and β‐Actin, respectively. It is presented as mean ± SEM. *** p < 0.001 vs. Ctl.

Article Snippet: After blocking with 5% skim milk in TBST for 2 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against β actin (1:5000, A5441, Sigma), ErbB2 pY‐1248 (1:1000, AF1768, R&D Systems), ErbB2 (1:400, sc‐377,344, Santa Cruz Biotechnology), and ErbB1 (1:400, 2232S, Cell Signaling Technology).

Techniques: Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: ErbB2 pY ‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

doi: 10.1111/cns.14407

Figure Lengend Snippet: Western blot analysis of ErbB2 pY‐1248 in mouse PD model. (A) Representative images of western blot of ErbB2 pY‐1248 in the mouse brain tissues. (B) Grayscale value analysis of the immunoblots. The band intensity of ErbB2 pY‐1248 was normalized to β‐Actin. *** p < 0.001 vs. Ctl.

Article Snippet: After blocking with 5% skim milk in TBST for 2 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against β actin (1:5000, A5441, Sigma), ErbB2 pY‐1248 (1:1000, AF1768, R&D Systems), ErbB2 (1:400, sc‐377,344, Santa Cruz Biotechnology), and ErbB1 (1:400, 2232S, Cell Signaling Technology).

Techniques: Western Blot

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