Rabbit anti hsp60

5 × 10⁵ cells were collected, lysed using RIPA Cell lysis buffer (Thermo, Cat no. 89900), dosed using Pierce™ BCA Protein Assay kit (Thermo, Cat no. 23225), and diluted in 5X SDS‐page loading buffer (10% SDS, 500 mmol/L DTT, 50% Glycerol, 250 mmol/L Tris‐HCL and 0.5% bromophenol blue dye, pH = 6.8) at 1 µg/µL. 20 µg of protein extract was loaded onto a 10% SDS‐page gel, and migration was performed at 100 V for 90 minutes. After SDS‐PAGE electrophoresis, proteins were transferred onto a PVDF membrane (Biorad). Membranes were saturated with 5% (w/v) fat‐free dry milk, then probed with primary antibodies: mouse anti‐Cas9 (EpiGentek, Cat no. A‐9000), rabbit anti‐HSP60 (Santa Cruz, Cat no. SC13115), rabbit anti‐BIM (Sigma‐Aldrich, Cat no. B7929). All antibodies were used at a 1/1000 dilution. After secondary antibody labeling, peroxidase activity was revealed using Western Lightning Plus‐ECL kit (Perkin Elmer, Cat no. NEL103001EA) and Kodac X‐ray films (abm, Cat no. B503).

Antibodies were obtained from the following sources: goat anti-C. trachomatis MOMP (Meridian Life Sciences, Saco, ME, USA), rhodamine red X conjugated anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA), HRP conjugated anti-rabbit secondary antibody (Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-HSP60 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HRP conjugated anti-mouse secondary antibody (Cell Signaling Technologies, Danvers, MA, USA). Fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA), Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Grand Island, NY, USA) and phosphate buffered saline (PBS) were purchased from (Lonza, Walkersville, MD, USA). Ethyl acetate, ammonium formate, and dichloromethane were obtained from ACROS Organics (Pittsburgh, PA, USA) and 3,3′-diindolylmethane was from Santa Cruz Biotechnology, Santa Cruz, CA. Acetonitrile and water were HPLC grade from Fisher Scientific and Sep-Pak Vac 3cc (200mg) certified silica cartridges were purchased from Waters [22 (link)], part No: 186004614. Spilanthol was obtained from Santa Cruz Biotechnology (Dallas, TX, USA; sc-474036) and Spilanthol endoperoxide (SPL^E) was synthesized as described below.

Cell extracts were prepared as previously described [65 (link)] and analysed using the following primary antibodies: rabbit anti-Sox9 (Millipore, AB5535), rabbit anti-ALDH1A3 (Abgent, RB16818), mouse anti-GAPDH (Sigma, G8795), mouse anti-β-actin (AC-15/A5441), mouse anti-ERα (Novocastra Leica Biosystems, NCL-ER-6F11), Armenian hamster anti-Muc-1 (ThermoFisher Scientific, MA5-11202), mouse anti-β-tubulin (Sigma, T4026) and rabbit anti-Hsp60 (Santa Cruz Biotechnology, sc-13966). For detection an enhanced chemiluminescence detection kit (Bio-rad) was used.