Anti mouse igg secondary antibody

MSC or CAF cells were harvested in a radioimmunoprecipitation assay buffer for western blotting analysis (Beyotime, Haimen, Jiangsu, China). Mouse anti-collagen I monoclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-collagen III polyclonal antibody (Abcam), rabbit anti-FAP polyclonal antibody (Abnova, Taipei, Taiwan), rabbit anti-α-SMA monoclonal antibody (Epitomics, Burlingame, CA,USA), rabbit anti-GDF15 polyclonal antibody (Bioworld, Louis Park, MN,USA) and mouse anti-β-actin monoclonal monoclonal antibody (Cell Signaling, Danvers, MA, USA) were used. Antigen detection was performed using a goat anti-rabbit or an anti-mouse IgG secondary antibody (Cell Signaling) conjugated to HRP and an ECL western blotting substrate (Pierce, Waltham, MA, USA). The BM aspirates were collected from newly diagnosed AML patients which were divided into CR group (n = 9) and refractory group (n = 8) and normal controls (n = 11). The ELISA analysis for GDF15 was performed according to the manufacturer’s instructions (R&D systems).

To assess LC3-I/II, p62/SQSTM1, HSPA5 and GAPDH expression, western blot analysis was carried out on 10% or 15% SDS-PAGE as previously described [38 (link)]. The anti-LC3 and anti-GAPDH antibodies were purchased from Medical & Biological Laboratories (MBL, Japan). The anti-HSPA5 antibody was purchased from Abcam (Cambridge, UK). The anti-p62/SQSTM1 antibody was purchased from Abways Technology (Shanghai, China). Anti-rabbit IgG secondary antibody and anti-mouse IgG secondary antibody were purchased from Cell Signaling (Danvers, USA). The dilution ratio of all antibodies was 1:1000. Band intensities were quantified using ImageJ software (NIH, USA).

After 24 hours culture under normoxia or hypoxia, 1 × 10⁶ cells of NB4 or THP-1 were lysed in 0.1 ml Blue Loading Buffer Pack (Cell Signaling #7722, Danvers, MA) and 0.02 ml of lysed cells were electrophoresed in 7.5% polyacrylamide gel. The gel was electroblotted onto hybond-P (GE Healthcare, UK). The membrane was hybridized with antibody to PDK1 (mouse monoclonal antibody, IgG1: abcam #ab110335, UK) at 1:1000 dilution and reacted with anti-mouse IgG secondary antibody (Cell Signaling #7076, Danvers, MA) at 1:2000 dilution. The membrane was visualized by ECL-prime Detection System (GE Healthcare, UK). β-actin (horseradish peroxidase-conjugated rabbit monoclonal antibody, IgG: Cell Signaling #5125, Danvers, MA) was also tested as control.

Cells and tissues were ground and lysed using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 1% sodium deoxycholate, and 5 mM EDTA). Samples were loaded and separated on 10–12% SDS polyacrylamide gels and transferred onto PROTRAN nitrocellulose membranes (Schleicher and Schuell Co., Keene, NH, USA). After blocking with PBS containing 5% non-fat dry skim milk and 0.07% (vol/vol) Tween 20, membranes were incubated with an antibody against β-catenin (1:1000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), α-smooth muscle actin (1:1000; α-SMA, Abcam, Cambridge, UK), Keratin 14 (1:1000; Covance, Burlington, NC, USA), Collagen I (1:1000; Abcam), proliferating cell nuclear antigen (1:500; PCNA, Santa Cruz Biotechnology), α-tubulin (1:5000; Oncogene Research Products, La Jolla, CA, USA), or Erk (1:5000; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. Samples were then incubated with horseradish peroxidase–conjugated anti-rabbit (1:5000; Bio-Rad Laboratories, Hercules, CA, USA) or anti-mouse IgG secondary antibody (1:5000; Cell Signaling Technology). Protein bands were visualized using an enhanced chemiluminescence kit (Amersham Bioscience, Piscataway, NJ, USA) and a luminescent image analyzer, LAS-4000 (Fujifilm, Tokyo, Japan).

Nectin-4-MMAE is a gift from Mabwell Bioscience CO., LTD. Shanghai China. Autophagy modulators: LY294002 (Medchemexpress, HY-10108, Shanghai China); Chloroquine (Sigma-Aldrich, C6628, Darmstadt Germany); Rapamycin (Sangon Biotech, A606203, Shanghai China). Cell viability assay: MTT (Beyotime, C0009S, Shanghai, China). ADC labeling: AlexaFlour 488 protein label kit (ThermoFisher, A10235, MA US). Cell apoptosis assay: cell apoptosis detection kit (Meilunbio MA0220-1, Dalian China). Autophagy assay: Cyto-ID autophagy detection kit (Enzo Life Sciences, ENZO-51031-K200, NY US); Lyso-Tracker-DND 99 (Invitrogen, L7528, CA US). Antibodies: Primary antibody: anti-GAPDH, #2118; anti-LC3 I/II, #3868; anti-SQSTM1, #8025; anti-caspase 3, #9662; anti-PARP, #9542; anti-phospho-mTOR (Ser2448), #2971; anti-mTOR, #2983; anti-phospho-AKT (Ser473), #4060; anti-phospho-p70s6 Kinase (Ser371), #9208; anti-p70s6 Kinase, #2708; anti-phospho-4EBP1 (Thr45), #2971; anti-4EBP1, #9644, anti-PI3 Kinase, #4257; anti-phospho-PI3 Kinase, #4228 (Cell Signaling Technology, MA US). Anti-PDK-1, AF7707 (Beyotime Biotechnology, Shanghai, China). Anti-PTEN, A19104 (Abclonal, Wuhan China). Secondary antibody: HRP-conjugated anti-rabbit secondary antibody, #7074; anti-mouse IgG secondary antibody, #7076 (Cell Signaling Technology, MA US).