Ecl western blotting detection kit

The western blot experiment was carried out according to the description [14 (link)]. After separation by SDS-PAGE, the designated proteins were transferred to the PVDF membranes, and the PVDF membranes were horizontally cut based on the location of the target molecule. The following antibodies were used in this research: anti-DGKA (CA60796, 1:1000, Cell Signaling Technology, MA, USA), anti-Jak1 (CA29261, CST), anti-Jak3 (CA8863, CST), anti-Stat5 (CA25656, CST), anti-P-Stat5 (Tyr694) (CA72712, CST), anti-Stat3 (CA12640, CST), anti-P-Stat3 (Tyr705) (CA9145, CST), anti-PARP (CA9532, CST), anti-cleaved PARP (CA5625, CST), anti-caspase-3 (CA9662, CST), anti-MEK (CA8727, CST), anti-P-MEK (CA3958, CST), anti-ERK (CA4695, CST), anti-P-ERK (CA8544, CST), anti-GAPDH (CA5174, CST), anti-SphK1 (SP5421, 1:1000, ECM Biosciences, KY, USA), anti-P-SphK1 (Ser-225) (SP1641, ECM Biosciences) and HRP-linked anti-rabbit IgG (CA7074, CST). Finally, all protein were visualized by ECL Western blotting Detection Kit (GeneFlow, Staffordshire, UK). The protein agonists applied were as follows: SphK1 agonist K6PC-5(HY-124042, MedChemExpress); MEK/ERK agonists C16-PAF (HY-108635, MedChemExpress); JAK/STAT agonists RO8191 (T22142, TargetMol).

2×10⁵/well cells were treated with chidamide in the absence or presence of and/or MI-3 for 48 h, and the subjected to western blot analysis using indicated primary antibodies and secondary HRP-conjugated antibodies (1:10,000, Abcam, Cambridge, UK). The primary antibodies included anti-caspase-3 (#9662S), anti-PARP (#9532S), anti-histone H3 (#4499S), anti-phospho-H3 (#53348S), anti-γH2A.X (#2577S), anti-RAD51 (#8875S), anti-KU70 (#4588S), anti-STAT3 (#9139 S), anti-Mcl-1 (#94296S), anti-phospho-p53 (#9286S), anti-p21(#2947S), anti-phospho-ATM (#5803S), anti-ATM (#2873S), anti-phospho-ATR (#2835S), anti-CHK1 (#2360), anti-CHK2 (#2662), anti-P-CHK1 (#2197S), and anti-P-CHK2 (#2348S) from Cell Signaling Technology (Boston, MA, USA). The primary antibodies were diluted with 5% fat-free milk-TBST. Anti-β-actin (1:1000, Cell Signaling Technology) was used as loading control. Blots were then detected using the ECL Western Blotting Detection Kit (GeneFlow, Staffordshire, UK).

The protein expression levels were determined by staining with primary antibodies and HRP conjugated secondary (1:5000, Armersham, Buckinghamshire, UK) antibody. The cleaved PARP, BCL2, BAX (1:1000, Abcam, Cambridge, UK) and anti-α-tubulin (1:8000, Sigma) primary antibodies were diluted in 5% fat-free milk-TBST. The signal was detected using an ECL Western blotting detection kit (GeneFlow, Staffordshire, UK).

After treatment with anlotinib for the designated times, the cells were lysed, and the protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) based on the methods previously reported25 (link). Whole cell lysates (50 μg for each sample) were subjected to western blot analysis using the indicated primary antibodies and secondary HRP-conjugated antibodies (CA7074, 1:1000, Cell Signaling Technology, MA, USA). Blots were detected by visualization using the ECL Western Blotting Detection Kit (GeneFlow, Staffordshire, UK). The primary antibodies used were anti-β-actin (CA4970S, 1:1000, Cell Signaling Technology, MA, USA), anti-BAX (CA5023S, 1:1000, CST), anti-MCL1 (CA2538S, 1:1000, CST), anti-BAK (CA12105 1:1000, CST), anti-MYC (CA5605S 1:1000, CST), anti-PARP (CA9532, 1:1000, CST), anti-cleaved PARP (CA5625, 1:1000, CST), anti-caspase-3 (CA9662, 1:1000, CST), anti-cleaved caspase-3 (CA9661, 1:1000, CST), anti-p-p53 (Ser15) (CA9286, 1:1000, CST), anti-MDM2 (CA86934, 1:1000, CST), anti-γH2A.X (CA2577s, 1:1000, CST), and anti-SETD1A (ab70378, Abcam, Cambridge, UK) antibodies.

After treatment, a total 5 × 10⁶/ml cells per condition were lysed and the protein concentration was determined using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Whole cell lysates (50 μg for each sample) were electrophoresed in 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies (1:1000 in 5% bovine serum albumin tris-buffered saline with polysorbate 20; TBS-T) overnight at 4 °C, followed by horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology; 1:10,000 in 5% fat-free milk TBS-T) for 1.5 h at room temperature. The primary antibodies against Bcl-2 (CA4223S), Mcl-1 (CA2538S), caspase 3 (CA9662S), caspase 9 (CA5340S), PARP (CA9532S), BIM (CA2933S), PUMA (CA3741S), BID (CA7382S), and β-actin (CA4970S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The blots were probed with anti-β-actin antibody (rabbit mAb 1:1000, Cell Signaling Technology) to ensure equal loading and transfer. The proteins were visualized using ECL Western Blotting Detection Kit (Gene-Flow, Staffordshire, UK).

The protein expression levels were determined by staining with primary antibodies and relevant HRP conjugated secondary (1:5000, Armersham, Buckinghamshire, UK) antibodies. The primary antibodies (Santa Cruz, CA, USA: Bcl2, Bax, p65, IκBα, nucleolin, ALDH1A1, 1A3, 3A1, 2 and cleaved PARP; Novus Bio, Cambridge, UK: HIF2; Abcam, Cambridge, UK: phosphorylated p65_S276; Cell Signaling, Herts, UK: AKT, phosphorylated AKT, Sox2, Oct4, JNK, phosphorylated JNK, cJun, phosphorylated cJun, phosphorylated p38, ERK) were diluted at 1:1000 in 5% fat-free milk-TBST. Anti-α-tubulin (1:8000, Sigma) and nucleolin were used as a loading control. The signal was detected using an ECL Western blotting detection kit (GeneFlow, Staffordshire, UK).

Cell protein was extracted with RIPA protein lysis buffer (Beyotime). MOLM-13 cells (2 × 10⁵/mL) were cultured with 20 μM Apatinib or 16 nM HHT alone or combined for 24 h and 48 h. Related antibodies included β-actin, VEGFR-2, p-VEGFR-2, PI3K, Akt, CyclinA2, CyclinD1, P21, and BCL-2 (rabbit, 1 : 1000, Cell Signaling Technology). Blots were tested by the addition of a horseradish peroxidase (HRP)-conjugated secondary antibody. Signals were visualized using the ECL Western blotting detection kit (Gene-Flow, Staffordshire, UK).