Hrp conjugated goat anti mouse igg secondary antibody

Gedatolisib (purity: 99.09%) was purchased from Selleck Chemicals (Houston, TX, USA). Paclitaxel (purity: 99.02%), doxorubicin (purity: 99.37%), vincristine (purity: 99.19%), mitoxantrone hydrochloride (purity: 99.9%), topotecan hydrochloride (purity: 100.0%), and cisplatin (purity: 99.62%) were purchased from Energy Chemicals (Shanghai, China). Verapamil hydrochloride (purity: 99.95%) and Ko143 (purity: 99.79%) were purchased from MedChemExpress (NJ, USA). Tritium-labeled paclitaxel and mitoxantrone were obtained from SuYa Medicine (Beijing, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s Medium (DMEM), penicillin/streptomycin, and trypsin were purchased from Corning Incorporated (Corning, NY, USA). G418, DMSO, 3-[4,5-dimethylthiazol-yl]-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and paraformaldehyde, were purchased from Beyotime Biotechnology (Shanghai, China). Antibody of GAPDH, ABCB1, and ABCG2, Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody, HRP-conjugated goat anti-mouse IgG secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were obtained from Sigma Chemical Co (St. Louis, MO, USA).

Briefly, MV4-11 cells were incubated
with inhibitors prior to cell lysis with radioimmunoprecipitation
assay (RIPA) buffer (20 mM Tris pH 7.4, 150 mM NaCl, 0.5% deoxycholate,
1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS)). Total protein
content was determined through a BCA assay (ThermoFisher), resolved
via a 4–20% polyacrylamide SDS gel, and transferred to a nitrocellulose
membrane (Bio-Rad). The membranes were blocked with a 5% solution
(skimmed milk powder in PBS-T). This was followed by incubation at
4 °C (overnight) with the following antibodies: acetylated α-tubulin
mouse monoclonal (MABT868, EMD Millipore), acetylated histone H3 (Ac-Lys18,
07–354, Sigma), PARP-1 (ab227244, Abcam), apoptosis Western
blot cocktail (136812, Abcam), cleaved PARP-1 (ab32561, Abcam), and
HSC70 (sc-7298, Santa Cruz). Following overnight incubation, horseradish
peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody
(7076, Cell Signaling) or HRP-linked anti-rabbit IgG secondary antibody
(7074, Cell Signaling) was applied to the membrane in a 1:5000 dilution.
The bands were visualized using clarity Western ECL substrate luminal/enhancer
solution and peroxide solution. Western blotting analysis was carried
out using Image lab software (Bio-Rad).^{47 (link),50 (link),70 (link)}

Equal amounts of SEA and SjHSP60-depleted SEA or SEA and SWA (80 μg) were loaded in each lane for separation by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking in Tris-buffered saline containing Tween 20 (0.1%) (T-TBS) and milk (5%) at room temperature for 2 h, the membrane was then incubated at 4°C overnight with anti-SjHSP60 mAb (S-129-5; 1:500 diluted). After washing with T-TBS, the membrane was incubated at room temperature for 1 h with HRP-conjugated goat anti-mouse IgG secondary antibody (Cell Signaling Technology, Danvers, MA). In parallel, Coomassie Blue staining was used to confirm that the equal amounts of antigens were used in Western blot analysis.