Journal: bioRxiv

Article Title: The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

doi: 10.1101/2022.02.08.479504

Figure Lengend Snippet: (A-C) Cells expressing either Ssa1, 2, 3 or 4 as their sole Ssa and HA-tagged Rnr1/Rnr2/Rnr4 were grown to exponential phase and were either left untreated or were treated with 200 mM HU for 3 hrs. HA-RNR complexes were immunoprecipitated with anti-HA magnetic beads and were subjected to SDS-PAGE and analyzed by immunoblotting with anti-HA antibodies to detect the RNR subunits or anti-Ydj1 antibodies to detect Ydj1. (D) Interaction between RNR and Ydj1 (Ydj1/RNR) was calculated by quantitating bands from three replicate experiment. Each value represents the mean ± SD (n = 3). Statistical significance between samples was calculated ANOVA. (∗∗∗∗ p < 0.01).

Article Snippet: Proteins were detected using the following antibodies; anti-HA tag (Thermo #26183), Anti-FLAG tag (Sigma, #F1365), anti-PGK1 (Thermo # PA5-28612), anti-Ydj1 (StressMarq #SMC-166D).

Techniques: Expressing, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot

Journal: Scientific Reports

Article Title: Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan

doi: 10.1038/srep28751

Figure Lengend Snippet: (a) Hsp90 levels are increased in the chaperone deficient mutants relative to the control. **p < 0.01 (ANOVA plus post hoc). (b) Oxygen consumption rates are decreased in the chaperone deficient mutants. Oxygen consumption is measured polarographically in the YPD growth medium supplemented with 2% glucose at 30 °C. (c) Mitochondrial membrane potential-related fluorescence via DiOC6(3) is decreased. After collapsing the ΔΨm by 10 min preincubation with 100 μM CCCP and 5 μg/mL antimycin, signal intensity decreased, indicating mitochondrial membrane depolarization. Mitochondrial biomass is unchanged in the chaperone deficient mutants. The NAO fluorescent signal of energized mitochondria was collected in the appropriate channel following staining. (d) ATP levels are decreased in the chaperone deficient mutants, without change in ROS levels. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p < 0.001; **p < 0.01 (ANOVA plus post hoc). (e) Drop test results display no difference in growth on YPEG medium between the studied strains. Growth on YPEG plates is preceded by growth in the same liquid medium. Left panel displays results of the exponentially growing cells, and the right one of the stationary cells. (f) Blue Native gels showing the level of Complexes II, IV and V in the wild type and the chaperone deficient strains.

Article Snippet: Anti-Hsp90 antibody (1 mg/mL) was diluted in blocking buffer (1:2500, StressMarq Biosciences), and incubated overnight at 4 °C.

Techniques: Fluorescence, Staining

Journal: Scientific Reports

Article Title: Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan

doi: 10.1038/srep28751

Figure Lengend Snippet: (a) Western blot analysis of several respiratory chain components. Porin was used as a loading control. (b) Representative images of cells with visualized mitochondria via preCOX4-mCherry and preSU9-GFP. (c) CORE pathway transcript level in the Coa3-null mutant as well as the wild type treated with 25 μM CCCP. (d) Hsp90 levels are unaffected by the CCCP treatment in the wild type strain. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p < 0.001; **p < 0.01 (ANOVA plus post hoc). (e) Venn diagram of the overlap between the responses in chaperone deficient strains, Coa3-null mutants, and the CCCP-treated wild type cells.

Article Snippet: Anti-Hsp90 antibody (1 mg/mL) was diluted in blocking buffer (1:2500, StressMarq Biosciences), and incubated overnight at 4 °C.

Techniques: Western Blot, Mutagenesis

Journal: Scientific Reports

Article Title: Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan

doi: 10.1038/srep28751

Figure Lengend Snippet: (a) Transcript levels of genes proposed to be involved in the CORE pathway. UBC6 was used as a control. The measurement was performed in biological and technical triplicate. (b) Hsp90 levels remain unaffected by the deletion of studied chaperones in the absence of Hsf1. Data are represented as mean ± SD from 3 independent cultures, each measured in duplicate. ***p < 0.001; **p < 0.01 (ANOVA plus post hoc).

Article Snippet: Anti-Hsp90 antibody (1 mg/mL) was diluted in blocking buffer (1:2500, StressMarq Biosciences), and incubated overnight at 4 °C.

Techniques:

Journal: Proteome Science

Article Title: PRM-based quantitative proteomics analysis of altered HSP abundance in villi and decidua of patients with early missed abortion

doi: 10.1186/s12953-023-00213-w

Figure Lengend Snippet: Quantitative and statistical analysis of target peptide in decidua of 10 patients with EMA and 10 controls identified using PRM-based targeted proteomics

Article Snippet: Human HSP90AB1 antibody (Boster, Wuhan, China, no. BM4191, 1:2000 dilution), human HSPD1 antibody (Boster, Wuhan, China, no. M01280-3, 1:2000 dilution) and human HSPA13 antibody (Proteintech, Wuhan, China, no. 12667–2-AP, 1:2000 dilution) were used as the primary antibodies for Western blot analysis.

Techniques: Sequencing, Control

Journal: Proteome Science

Article Title: PRM-based quantitative proteomics analysis of altered HSP abundance in villi and decidua of patients with early missed abortion

doi: 10.1186/s12953-023-00213-w

Figure Lengend Snippet: Quantitative and statistical analysis of target peptide in villi of 10 patients with EMA and 10 controls identified using PRM-based targeted proteomics

Article Snippet: Human HSP90AB1 antibody (Boster, Wuhan, China, no. BM4191, 1:2000 dilution), human HSPD1 antibody (Boster, Wuhan, China, no. M01280-3, 1:2000 dilution) and human HSPA13 antibody (Proteintech, Wuhan, China, no. 12667–2-AP, 1:2000 dilution) were used as the primary antibodies for Western blot analysis.

Techniques: Sequencing, Control

Journal: Proteome Science

Article Title: PRM-based quantitative proteomics analysis of altered HSP abundance in villi and decidua of patients with early missed abortion

doi: 10.1186/s12953-023-00213-w

Figure Lengend Snippet: Differentially expressed HSPs proteins in decidua of 10 patients with EMA and 10 controls identified using PRM-based targeted proteomics

Article Snippet: Human HSP90AB1 antibody (Boster, Wuhan, China, no. BM4191, 1:2000 dilution), human HSPD1 antibody (Boster, Wuhan, China, no. M01280-3, 1:2000 dilution) and human HSPA13 antibody (Proteintech, Wuhan, China, no. 12667–2-AP, 1:2000 dilution) were used as the primary antibodies for Western blot analysis.

Techniques: Membrane

Journal: Proteome Science

Article Title: PRM-based quantitative proteomics analysis of altered HSP abundance in villi and decidua of patients with early missed abortion

doi: 10.1186/s12953-023-00213-w

Figure Lengend Snippet: Differentially expressed HSPs proteins in villi of 10 patients with EMA and 10 controls identified using PRM-based targeted proteomics

Article Snippet: Human HSP90AB1 antibody (Boster, Wuhan, China, no. BM4191, 1:2000 dilution), human HSPD1 antibody (Boster, Wuhan, China, no. M01280-3, 1:2000 dilution) and human HSPA13 antibody (Proteintech, Wuhan, China, no. 12667–2-AP, 1:2000 dilution) were used as the primary antibodies for Western blot analysis.

Techniques: Membrane

Journal: Proteome Science

Article Title: PRM-based quantitative proteomics analysis of altered HSP abundance in villi and decidua of patients with early missed abortion

doi: 10.1186/s12953-023-00213-w

Figure Lengend Snippet: The differential abundance of HSP90AB1, HSPD1 and HSPA13 in villi tissues was verified by Western blot. A The representative of Western blot analysis to verify selected differentially expressed proteins HSP90AB1, HSPD1 and HSPA13 in the villi tissue of embryo from EMA ( n = 6) and control( n = 6). B The scatter plot of HSP90AB1 abundance in the villi tissue of embryo from EMA ( n = 22) vs control ( n = 22) ( P < 0.01). C The scatter plot of HSPD1 abundance in the villi tissue of embryo from EMA ( n = 22) vs control ( n = 22) ( P < 0.01). D The scatter plot of HSPA13 abundance in the villi tissue of embryo from EMA ( n = 22) vs control ( n = 22) ( P < 0.01)

Article Snippet: Human HSP90AB1 antibody (Boster, Wuhan, China, no. BM4191, 1:2000 dilution), human HSPD1 antibody (Boster, Wuhan, China, no. M01280-3, 1:2000 dilution) and human HSPA13 antibody (Proteintech, Wuhan, China, no. 12667–2-AP, 1:2000 dilution) were used as the primary antibodies for Western blot analysis.

Techniques: Western Blot, Control

Journal: Proteome Science

Article Title: PRM-based quantitative proteomics analysis of altered HSP abundance in villi and decidua of patients with early missed abortion

doi: 10.1186/s12953-023-00213-w

Figure Lengend Snippet: A , B , C Immunohistochemical staining for HSP90AB1, HSPD1, and HSPA13 observed in the cytoplasm of syncytiotrophoblast and cytotrophoblast cells (× 200, × 400). Villus from 22 patients with EMA showed lower; D - F Quantitative scoring results of IHC analyses w shown as box plots

Article Snippet: Human HSP90AB1 antibody (Boster, Wuhan, China, no. BM4191, 1:2000 dilution), human HSPD1 antibody (Boster, Wuhan, China, no. M01280-3, 1:2000 dilution) and human HSPA13 antibody (Proteintech, Wuhan, China, no. 12667–2-AP, 1:2000 dilution) were used as the primary antibodies for Western blot analysis.

Techniques: Immunohistochemical staining, Staining

Journal: Frontiers in Plant Science

Article Title: Novel α-Tubulin Mutations Conferring Resistance to Dinitroaniline Herbicides in Lolium rigidum

doi: 10.3389/fpls.2018.00097

Figure Lengend Snippet: Western blot analysis of expression of WT, and mutant 243-Met and 243-Lys α-tubulin variants in transgenic rice seedlings in comparison to an untransformed control, probed by antibody against fused HA (hemagglutinin) epitope tag. HSP (Heat shock protein 70) expression was used as a protein loading control.

Article Snippet: The membrane was blocked in TBST buffer containing 5% (w/v) milk powder and 0.1% Triton X-100, washed with fresh TBST buffer, and probed with a 1:10,000 dilution of mouse anti-heat-shock protein 70 (HSP) or rabbit anti-HA primary antibody (TransGen Biotech, China).

Techniques: Western Blot, Expressing, Mutagenesis, Transgenic Assay