Anti cyclin b1

Human glioblastoma cells or tumor tissues were harvested and washed with ice-cold PBS. Then, samples were suspended in SDS sample buffer, boiled for 10 min, and centrifuged at 12,000 rpm for 15 min. Thirty micrograms of protein samples were separated using 8–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore Corporation). The PVDF membrane was blocked with 5% milk for 1 h and then incubated with a primary antibody at 4°C overnight. Subsequently, the PVDF membrane was probed with the secondary antibody and visualized by enhanced chemiluminescence (ECL) (Beyotime Institute of Biotechnology, Haimen, China). The primary antibodies were as follows: anti-G9a (1:500, Santa Cruz), anti-a-tubulin (1:2000, Sigma), anti-CDK1 (1:1000, Abcam Cambridge, MA, United States), anti-CDK2 (1:500, Santa Cruz), anti-Cyclin A2 (1:2000, Abcam), anti-Cyclin B1 (1:3000, Abcam), anti-MMP1 (1:1000, Abcam), anti-N-cadherin (1:5000, Abcam), anti-LC3B (1:1000, Cell Signaling Technologies, Danvers, MA, United States), anti-p62 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), and anti-H3K9me2 (1:2000, Abcam). Horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000), goat anti-rabbit IgG (1:20,000), and rabbit anti-goat IgG (1:10000) (KPL, Gaithersburg, Maryland, United States) were used as secondary antibodies.

Endometrial tissues from three RIF subjects and fifteen various staged controls were examined. Immunostaining was performed as described previously24 (link). The endometrial tissues were fixed in 4% paraformaldehyde (Sigma) and then embedded in paraffin. After deparaffinization, rehydration, antigen retrieval, inhibition of endogenous peroxidase activity, and blocking, sections were incubated with anti-FoxM1 antibody (1:500, Santa Cruz) overnight. HESCs cultured in chamber slides were fixed in 4% paraformaldehyde followed by permeabilization and blocking. Incubations with the respective primary antibodies including anti-FoxM1 (1:200, Santa Cruz), anti-cyclin B1 (1:600, Abcam) and anti-phospho-histone 3 (pH3, 1:200, CST) were performed overnight at 4°C. Fluorescence (cyanine 3)-conjugated secondary antibodies were used respectively to visualize the signal and nuclei were stained with DAPI (1 μg/ml, Sigma).

The total protein in transfected KYSE450 and KYSE510 cells was extracted and quantified with the RIPA buffer (Solarbio, China), which was supplemented with 1% proteinase inhibitor cocktail. The protein concentration was obtained with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). Next, the quantified protein was loaded into 10% or 12% SDS-PAGE for electrophoresis separation. The separated protein bands were then electronically transferred to the membranes (Sigma-Aldrich, USA). Following that, the membranes were put under ice for 2 h with 5% BSA at room temperature. This step was followed by the incubation of the membranes overnight with primary antibodies at 4 °C and 1.5 h incubation with secondary detection antibody (Cat# ab205718, Abcam, USA) at room temperature. Finally, the protein immunoblots were visualized using the Enhanced Chemiluminescent (ECL) Reagent Kit (Thermo Fisher Scientific. USA). The density of the blots was obtained using ImageJ software. All the primary antibodies were purchased from Abcam (USA), including anti-CyclinB1 (Cat# ab32053), anti-ICAM1 (Cat# ab53013), anti-VCAM1 (Cat# ab134047), anti-Cleaved PARP (Cat# ab32561), anti-Bax (Cat# ab32503), anti-Cleaved Caspase-3 (Cat# ab2302), anti-TXNRD1 (Cat# ab124954) and anti-β-actin (Cat# ab8227).

Proteins were extracted from tissues and cells using the HEPES lysis buffer (Invitrogen) and then boiled with SDS/PAGE loading buffer (Invitrogen) for protein denaturation. Proteins were separated using SDS/PAGE, followed by transferring on to nitrocellulose filter membrane. Target proteins in the NC membrane were examined via immunoblotting using the following primary antibodies: anti-DRG1 (Abcam, Cambridge, U.K.) anti-Cyclin B1 (Abcam), anti-CDK1 (Abcam), and anti-GAPDH (Abcam) as well as appropriate secondary antibodies. Protein level was quantified using a gel documentation and analysis system. GAPDH was used as the internal control to verify basal expression levels and equal protein loading.

Quercetin, AG490, IL‐6 and DMSO were obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS), trypsin, and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA). Anti‐N‐cadherin, anti‐Vimentin, anti‐E‐cadherin, anti‐MMP‐9, and anti‐Cyclin B1 antibodies were purchased from Abcam (Cambridge, UK). Anti‐Bax, anti‐LC3, anti‐Beclin1, anti‐PCNA and anti‐STAT3, anti‐phospho‐STAT3, anti‐total‐JAK2, anti‐phospho‐JAK2 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Antibody to anti‐P62 was obtained from Proteintech (Chicago, IL).

Proteins were extracted with Radio Immunoprecipitation Assay lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Beyotime, China). Protein samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subjected to western blot analysis using the following primary antibodies: anti‐PLK4 (1:500; Abcam), anti‐p53 (1:1000), anti‐phospho‐p53^Ser15 (1:1000), anti‐Cyclin B1 (1:1000), anti‐Cyclin D1 (1:1000), anti‐caspase‐3 (1:1000), anti‐cleaved caspase‐3 (1:1000), anti‐caspase‐9 (1:1000), anti‐cleaved caspase‐9 (1:1000), and anti‐β‐tubulin (1:1000). Then, the membranes were incubated with secondary antibodies (1:1000; all from Cell Signaling Technology). The protein bands were eventually visualized using an enhanced chemiluminescence detection kit (Millipore). The comparison of PLK4 expression between keloid dermis and adjacent normal skin dermis was made based on 21 biological replicates, while other western blot assays were conducted on at least three biological replicates.