Rabbit anti hsf1 antibody

Protein isolation and western blots were performed as described before [48 (link)]. For analysis of phosphorylated proteins, cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (89900, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (cOmpleteTM Tablets, 11697498001, Roche, Basel, Swiss; Sodium orthovanadate, 13721-39-6, Fivephoton Biochemicals, San Diego, CA, USA). Antibodies against rabbit anti-GLI1 monoclonal antibody (C68H3, 1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-GLI-2 (Dilution, C-10: sc-271786, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Anti-HSF1-phospho(S326) antibody (1:5000, EP1713Y, abcam, Cambridge, UK), rabbit Anti-HSF1 antibody (1:1000, 4356, Cell Signaling Technology, Inc., Danvers, MA, USA), Mouse anti-HSP70/HSPA1A antibody (1:1000, MAB1663, R&D Systems, Inc. Minneapolis, MN, USA), Rabbit anti-HSP90 Antibody (1:1000, #4874, Cell Signaling Technology, Inc., Danvers, MA, USA) or mouse anti-β-ACTIN (sc-47778, 1:5000, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies against HRP-linked anti-rabbit immunoglobulins (1:10,000, 7074) and anti-mouse IgG (1:10,000, NXA931) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and GE Healthcare (Cytiva, Marlborough, MA, USA), respectively.

Eight-week-old, end-stage TDP-43
^WTxQ331Kmice and age-matched littermates were anaesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in phosphate buffer. Spinal cord was postfixed in 4% PFA in 15% sucrose for 5 h, cryoprotected in 30% sucrose for 24 h and cut into 30 µm sections on a cryostat. Sections were incubated in rabbit anti-HSF1 antibody (1:250; Cell Signaling), washed and incubated with a biotinylated anti-rabbit antibody (1:1000; Vector) and then incubated in an ABC kit (Vector). Sections were imaged using a Zeiss light microscope and Axiovision software.

NT2 cells grown overnight on coverslips (1 × 10⁵ cells/ml density) in presence of dialyzed fetal bovine serum were fixed in 4% paraformaldehyde in PBS at RT for 10 min. Fixed cells were permeabilized with 0.1% Triton-X-100 in PBS at 37°C for 5 min, blocked with 1% BSA in PBS at 37°C for 30 min, and incubated with rabbit anti-HSF1 antibody (1:100 dilution) (catalog no. 4356, RRID:AB_2120258, Cell Signaling Technology) for 2 hr. After washing, cells were incubated with AlexaFluor 488-conjugated goat anti-rabbit IgG (H+L) (catalog no. A-11008, RRID:AB_143165, Invitrogen) for 2 hr. After washing, coverslips were mounted in Vectashield mounting medium containing DAPI and imaged as mentioned earlier. Images were collected using a Leica Confocal SPE8 microscope using a 63 × numerical aperture 1.42 oil-immersion objective lens. The relative intensity of HSF1 in the nuclei of control NT2 cells, and cells treated with BIMU8 in the presence or absence of H89 was quantified from the projections of confocal z-stacks using ImageJ. Background signal was subtracted from each of the projections and the mean intensity for regions corresponding to nuclei of the cells was determined. The average of 25 cells was used to determine the mean intensity for HSF1 staining.