Anti hsp90

Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).

Untreated mouse WT and IL17A-depleted M1- and M2-like macrophages, or those treated with gemcitabine, were collected and resuspended in lysis buffer (TBS, 0.5% Triton X-100, 0.5% NP-40, 1mM PMSF, 1mM Na₃VO₄, 1% DTT and protease inhibitor cocktail). Protein concentration was evaluated using the Bio-Rad Protein Assay (Bio-Rad). Samples were resuspended in 4× Bolt LDS Sample Buffer and 10× reducing agent, and incubated at 70 °C for 10 min before loading on precast Bolt 4–12% Bis-Tris Plus gels (all from Thermo Fisher Scientific, Monza, Italy). Immunoblotting was performed with the following primary Abs: anti-NOS2 (Cell Signaling, supplied by Euroclone, Pero Mi, Italy), anti-ARG1 (Cell Signaling) and anti-HSP90 (Cell Signaling) following the manufacturer’s instructions. Anti–rabbit HRP-conjugated secondary Abs (diluted 1:10,000) were obtained from Thermo Fisher Scientific. Membranes were developed for chemiluminescent detection, and images were acquired using a ChemiDoc MP Imaging System and Image Lab 6.0.1 Software (Bio-Rad). Immunoblots were quantified by ImageJ 1.45 software (National Institutes of Health, Bethesda, MD, USA).

Cells were washed once with ice cold PBS on ice, then lysed in 2x Laemmli Sample buffer, scraped and sonicated. All of the samples were boiled for 6 minutes at 100°C and analyzed by Western blotting. Protein samples were separated by 10% SDS-PAGE and transferred to 0.20 µM pore size nitrocellulose membrane with Trans-Blot^® Turbo™ Transfer System, 1.3 A, 25V for 10 minutes. The membranes were then blocked with 5% (w/v) nonfat dry-milk powder solution in TBS-Tween 20 (TBST) and incubated with specific primary antibodies overnight at 4°C. The primary antibodies used in this study were as follows: anti-Hsp90 (Cell Signaling Technology, Cat# 4874), anti-caveolin-1 (Cell Signaling Technology Cat# 3238), β-tubulin (BD Biosciences, Cat# 556321), anti-dynamin II (BD Biosciences, Cat# 610263). After incubation with the primary antibodies, the membranes were washed three times for 10 minutes with TBST and incubated with horseradish-peroxidase (HRP) conjugated anti-mouse (Cell Signaling Technology, Cat# 7076) or anti-rabbit (Cell Signaling Technology, Cat# 7074) secondary antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) using autoradiography films. ImageJ software (Research Services Branch, National Institute of Health (Bethesda, MD) was used for densitometric analyses.

The murine colon carcinoma cell line, CT26, murine mammary carcinoma cell line, 4T1 (gift of Yang-Xin Fu), and three human colon cancer cell lines HCT116, SW480, and HT29 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) where each lot was STR profiled. All cell lines were authenticated either by ATCC or IDEXX (4T1), tested for mycoplasma, and used at low passage numbers within 6 months. Cells were grown in the following media: RPMI-1640 (CT26); McCoy's 5A (HCT116); minimum essential medium Eagle (SW480); and Dulbecco's modified Eagle's mMedium (HT29). All media were supplemented with 10% FCS, 2 mM glutamine, and penicillin–streptomycin antibiotic, and the cells were incubated at 37 °C in 5% CO₂. RB solution of 10% was provided by Provectus Biopharmaceuticals (Knoxville, TN, USA). Annexin V-Biotin Apoptosis Detection Kit, streptavidin-FITC, and streptavidin-APC were purchased from eBioscience (San Diego, CA, USA). Anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CRT (Abcam, Cambridge, MA, USA), anti-HSP90, and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) antibodies were purchased and used according to the manufacturer's specifications. DAPI, quinacrin dihydrochloride, and chloroquine diphosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA).