Journal: PLoS ONE

Article Title: An Atypical Unfolded Protein Response in Heat Shocked Cells

doi: 10.1371/journal.pone.0023512

Figure Lengend Snippet: (A) Expression of dnHSF1. HEK-dnHSF1 were cultured in the absence or presence of doxycycline to induce expression of dnHSF1. After harvesting the cells were subjected to SDS-PAGE and levels of endogeneous HSF1 and dnHSF1 were determined by western blotting with β-actin as a loading control. (B) The effects of dnHSF1 on basal and heat shock induced activity of various promoters. Cells were transfected with a mixture of the indicated luciferase reporter and a β -actin-β-galactosidase reporter (9∶1 ratio). At 24 hours after transfection doxycyclin was added to induce expression of dnHSF1. At 48 hours after transfection, cells were exposed to a heat shock of 30′ at 45°C or left at 37°C. When heat shocked, cells were allowed to recover for the indicated periods at 37°C. Harvested cells were assayed for reporter gene activities. (C) Expression of dpHSF1 leads to increased HSP levels. HEK-cDNA5 cells were tranfected with pcDNA5-dpHSF1. At 24 hours after transfection cells were cultured in the absence or presence of doxycycline to induce expression of dpHSF1. At 48 hours after transfection cells were harvested and subjected to SDS-PAGE and levels of endogenous HSF1, dpHSF1, HSPA1A, DNAJB1 and HSPB1 were determined by western blotting. (D) The effect of dpHSF1 on the activity of various promoters. HEK-cDNA5 cells were transfected with a mixture (4∶1∶5) of the indicated luciferase reporter, a βactin-βgal reporter, and pcDNA5-dpHSF1. At 24 hours after transfection doxycycline was added to induce expression of dpHSF1. At 48 hours after transfection, cells were harvested and assayed for reporter gene activities.

Article Snippet: For western blot analysis, polyclonal HSF1 antibody (SPA-901; Stressgen) was used at a 1∶15,000 dilution, HSPA1A antibody 4G4 (ab5444; Abcam) was used at a 1∶5,000 dilution, polyclonal HSPA5 antibody, kindly donated by Prof. Dr. Ineke Braakman, was used at a dilution of 1∶1000, polyclonal DNAJB1 antibody (anti-Hsp40; SPA-400; Stressgen) at a 1∶10,000 dilution, HSPB1 antibody, obtained from Dr. A. Zantema, at a dilution of 1∶400, polyclonal XBP1 antibody [(M-186): sc-7160; Santa Cruz Biotechnology], at a dilution of 1∶200, polyclonal ATF4 antibody [(C-20): sc-200; Santa Cruz Biotechnology], at a dilution of 1∶1000, monoclonal eIF2α antibody was at a 1∶500 dilution, polyclonal phosphorylated eIF2α antibody (E2152; Sigma) was used at a 1∶1,000 dilution, monoclonal γ-tubulin antibody (GTU-88; Abcam) at 1∶1000 dilution and monoclonal β-actin antibody (AC-15, Sigma-Aldrich) at a dilution of 1∶5,000.

Techniques: Expressing, Cell Culture, SDS Page, Western Blot, Activity Assay, Transfection, Luciferase

Journal: PLoS ONE

Article Title: An Atypical Unfolded Protein Response in Heat Shocked Cells

doi: 10.1371/journal.pone.0023512

Figure Lengend Snippet: Oligonucleotides.

Article Snippet: For western blot analysis, polyclonal HSF1 antibody (SPA-901; Stressgen) was used at a 1∶15,000 dilution, HSPA1A antibody 4G4 (ab5444; Abcam) was used at a 1∶5,000 dilution, polyclonal HSPA5 antibody, kindly donated by Prof. Dr. Ineke Braakman, was used at a dilution of 1∶1000, polyclonal DNAJB1 antibody (anti-Hsp40; SPA-400; Stressgen) at a 1∶10,000 dilution, HSPB1 antibody, obtained from Dr. A. Zantema, at a dilution of 1∶400, polyclonal XBP1 antibody [(M-186): sc-7160; Santa Cruz Biotechnology], at a dilution of 1∶200, polyclonal ATF4 antibody [(C-20): sc-200; Santa Cruz Biotechnology], at a dilution of 1∶1000, monoclonal eIF2α antibody was at a 1∶500 dilution, polyclonal phosphorylated eIF2α antibody (E2152; Sigma) was used at a 1∶1,000 dilution, monoclonal γ-tubulin antibody (GTU-88; Abcam) at 1∶1000 dilution and monoclonal β-actin antibody (AC-15, Sigma-Aldrich) at a dilution of 1∶5,000.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Co-chaperones DNAJA1 and DNAJB6 are critical for regulation of polyglutamine aggregation

doi: 10.1038/s41598-020-65046-5

Figure Lengend Snippet: Western blot analysis displaying knock-out of DNAJ genes. KO of DNAJA1( A ) DNAJB1 ( B ) and DNAJB6 ( C ) were analysed by probing the membranes with anti-DNAJA1, anti-DNAJB1 and anti-DNAJB6 antibodies respectively.

Article Snippet: Primary antibodies used were anti-DNAJA1 (1:2000, Protein tech biosite; Cat#11713-1-AP), anti-DNAJB6 (1:2000, Abcam; Cat#ab198995), anti-HSP40/DNAJB1 (1:2000, Enzo Life Sciences; Cat#ADI-SPA-400-D), anti-β-actin (1:10000, Sigma-Aldrich; Cat#A3854).

Techniques: Western Blot, Knock-Out