Horseradish peroxidase conjugated goat anti mouse secondary antibody

Primary antibodies were obtained from the following companies: (1) TRIM66, PCNA, caspase7, caspase9, MMP2, MMP19, Twist11, CDH1, Snail1, Snail2, ZEB1 and TGF-β1, Abcam; (2) p-Smad4, Thermo Fisher; (3) p53, p-Smad2, Smad2, Smad4 and GAPDH, CST Biotech. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody or goat anti-rabbit secondary antibody was purchased from Beyotime.
Treated and untreated cells were lysed in RIPA lysis buffer with fresh added protease inhibitor cocktail (Sigma). Protein concentration was assayed using BCA protein assay (Thermo Fisher). Equal amounts of protein were then subjected to SDS gel electrophoresis. Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane and immunoblotted with the respective antibodies. Signals were developed by enhanced chemiluminescence (ECL, Millipore). Band intensities were measured using Image J (NIH) and normalized to GAPDH.

Western blotting was performed as described (58 (link)). The following primary antibodies were used: mouse anti-V5 (1:5,000; Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-tubulin (1:2,000; Solarbio, Beijing, China), rabbit Anti-GAPDH (1:2,000; Solarbio), and rabbit anti-Hypusine (EMD Millipore; 1:1,000) antibodies, rabbit anti-eIF5A (1:1,000; ABclonal), rabbit anti-DHPS (1:200; abcam), rabbit anti-DOHH (1:500; Sigma-Aldrich). Secondary antibodies used were Goat Anti-Mouse horseradish peroxidase-conjugated secondary antibody (1:2,000; Beyotime, Shanghai, China) and Goat Anti-Rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000). Anti-His (M1001020, Solarbio), Anti-pAKT473 (GB13012-3, Servicebio), Anti-pAKT308 (ab66134, Abcam), Anti-ATPaseβ (GL Binchem synthesis), Anti-Inx1(GL Binchem synthesis), Anti-Inx2 (GL Binchem synthesis), Anti-Inx3(GL Binchem synthesis), Anti-Inx4 (GL Binchem synthesis), Cleaved-caspase (WL02117, Wanleibio), Anti-GAPDH (M1000110, Solarbio), Anti-β Tubulin (AF1216, Beyotime), Goat Anti-Mouse (A0216, Beyotime), Goat Anti-Rabbit (A0208, Beyotime). Proteins were semi-quantified via densitometry using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Western blotting was performed as previously described [38 (link)]. Briefly, 50 µg of protein was loaded per sample. Total protein was isolated and analyzed by blotting using appropriate antibodies: mouse anti-Tubulin monoclonal antibody (1:2000; #AT819; Beyotime, Shanghai, China), anti-Ac-Lys antibody (1:2000; #ICP0380; ImmuneChem Pharmaceuticals); HRP-labeled goat anti-mouse IgG (H + L) (1:2000; #A0216; Beyotime, Shanghai, China); HRP-labeled goat anti-rabbit IgG (H + L) (1:2000; #A0208; Beyotime, Shanghai, China), anti-mouse V5 Tag monoclonal antibody (1:2000; #R96025; Invitrogen, CA, USA), goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000; #0216; Beyotime, Shanghai, China), anti-CypD antibody, which was a rabbit polyclonal antibody generated for CypD, cloned from M. bicoloratus hemocytes and expressed in Escherichia coli using the pET-32a-CypD plasmid (1:2000; Bioworld Technology, Inc., Nanjing, China). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to measure protein band density.

4T1 cells were inoculated into 6‐well plates at a cell density of 1.6 × 10⁵ per well for overnight growth. The culture medium was exchanged with 1.6 mL of fresh medium containing 0–100 µg mL⁻¹pAu Pd HSs. After 6 h of incubation, the cells were irradiated with 808 nm laser (0.5 W cm⁻²) for 10 min and cultured for another 18 h. The control group without NIR laser irradiation was incubated at 37 °C for 24 h under dark conditions. The cells were lysed and collected with a cell lysis buffer and centrifuged to measure the protein content in the supernatant using the Bradford method. By calculation, the protein in each sample was quantified to 20 µg and 10% SDS‐PAGE gel was prepared for electrophoresis and transferred to a PVDF membrane. After sealing, β‐actin (1:1000, Beyotime), HSP70 antibody (1:1000, abcam), Caspase‐1 antibody (1:1000, Proteintech), or HO‐1 monoclonal antibody (1:1000, abcam) was co‐incubated with membranes for 12 h. After being washed with TBST solution, goat anti‐mouse horseradish peroxidase‐conjugated secondary antibody (1:1000, Beyotime) was added to incubate with the membrane for 1 h. After being washed 3 times with TBST buffer, the membrane was visualized by Bio‐Rad imaging system.