Alpha tubulin

During the study, various antibodies were employed, including PELI1 (ab199336, Abcam) for Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and immunoprecipitation (IP), Cyclin E1 (11554–1-AP, Proteintech), Cyclin D1 (60186–1-Ig, Proteintech), CDK7 (#2916, CST), CDK4 (GB111602–100, Servicebio), p27 (25614–1-AP, Proteintech), E-cadherin (20874–1-AP, Proteintech), N-cadherin (22018–1-AP, Proteintech), Vimentin (10366–1-AP, Proteintech), Snail (10399–1-AP, Proteintech), Alpha Tubulin (66031–1-Ig, Proteintech), RPS3 (66046–1-Ig for WB/IHC/IF, 11990–1-AP for IP, Proteintech), Ki67 (27309–1-AP, Proteintech), PCNA (10205–2-AP, Proteintech), p53 (66031–1-Ig for WB, 10442–1-AP for IP/mIHC, Proteintech), Flag (66008–4-Ig, Proteintech), HA (66006–2-Ig, Proteintech), Myc (60003–2-Ig, Proteintech). Fluorescent secondary antibodies included mouse secondary antibody (SA00009–1, Proteintech) and rabbit secondary antibody (SA00013–4, Proteintech).
Chemical reagents employed in the experiment consisted of CHX (Aladdin, China), MG132 (Sigma, USA), four-color multiplex immunohistochemistry kit (Absin, abs50012, China), PI (Solarbio, China), DAPI (Solarbio, China), and D-Luciferin Potassium salt (PerkinElmer, USA).

Western blotting was conducted according to a previous method.³¹ Total cell protein lysates were extracted on ice using the protease inhibitor cocktail‐containing radioimmunoprecipitation assay buffer. Protein concentrations were detected with a Bio‐Rad protein assay. Then, 10–30 μg of total cell protein extracts were subjected to 10% SDS‐PAGE and electroblotted onto PVDF membranes (Millipore). The membranes were exposed to primary antibodies at 4°C overnight, followed by HRP‐linked anti‐mouse and anti‐rabbit antibodies for 2 h. Primary antibodies used were rabbit anti‐Birc5 polyclonal antibody (10508, Proteintech), mouse anti‐PD‐L1 monoclonal antibody (66248, Proteintech), mouse anti‐GAPDH (60004, Proteintech) and alpha‐Tubulin (66031, Proteintech).

Cell Counting Kit-8 assay was performed as described [46 (link)]. For colony formation assay, cells were seeded in 6-well plates at a density of 1000 cells per well. After 7 days, colonies were visualized by crystal violet staining.
Serum starvation was used to induce cell cycle synchronization roughly before cell cycle distribution was analyzed by flow cytometry as described [46 (link)]. For analyzing FAM83D expression in the cell cycle, AGS cells were synchronized at G0/G1 by double-thymidine block. Briefly, cells were treated with 2mM thymidine (Sigma, USA) for 20 h, followed by culturing in fresh media for 9 h. After that, cells were treated again with thymidine for 16 h and were released from the block by washing in PBS (phosphate-buffered saline) and culturing in fresh media. Samples were taken after block at 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours for flow cytometry analysis and Western blot analysis.
For the immunofluorescence analyses, tumor cells were incubated in 8-well glass slides (Millipore, USA) with primary antibodies against FAM83D (Bioss, CHINA), alpha-Tubulin (Proteintech, USA), followed by incubation with Goat anti-mouse IgG (H+L), FITC conjugate or Goat anti-rabbit IgG (H+L), TRITC conjugate (Proteintech, USA). DNA was stained with 406-diamidino-2-phenylindole.

Total protein was extracted from cultured cells using RIPA Lysis Buffer (Beyotime, #P0013B) and the protein concentration was determined by BCA kit (Genstar, #E162). The total protein was loaded onto a sodium dodecyl sulfate-polyacrylamide gel and ran at 120 volts for 2 hours. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane and treated with 5% skim milk TBST (50 mM Tris; 150 mM NaCl; 0.05% Tween 20) closed for one h. The PVDF membrane was then incubated at 4°C overnight with primary antibody and at room temperature with enzyme-labeled secondary antibody for 1 hour. Super-sensitive ECL chemiluminescent substrate (Biosharp, #BL520A) was used to reveal protein bands. The antibodies used are: ZO-1 (1:6000, Proteintech, # 21773–1-AP), HIF-1α (1: 6000, Proteintech, #20960–1-AP), Occludin(1:6000, Proteintech, # 27260–1-AP), alpha-tubulin (1:2000, Proteintech, #11224–1-AP), Goat Anti-Rabbit IgG (H + L)-HRP Conjugate(1:4000, Bio-Rad, #1706515). ImageJ is used for grayscale measurement.

Cells were seeded in 6-well plates and treated according to the experimental design. Total protein was obtained after cell lysis with RIPA lysis buffer (Beyotime, China), and cleared lysates were denatured by boiling for 10 min. Proteins were then separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (ABclonal, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with fast block buffer (Epizyme, China) for 20 min at room temperature. The membranes were then incubated overnight at 4°C with the following primary antibodies: AMPK (2532, Cell Signaling Technology), p-AMPK (2535, Cell Signaling Technology), Beclin1 (3495, Cell Signaling Technology), p62 (sc-28359, Santa Cruz Biotechnology), LC3 (14600-1-AP, Proteintech), and alpha-Tubulin (66031-1-Ig, Proteintech). After washing, the membranes were then incubated with horseradish peroxidase- (HRP-) conjugated goat anti-mouse and goat anti-rabbit antibodies (AS014 and AS003, ABclonal) for 1 h at room temperature.

RIPA (Weiao, China) was used to lyse harvested cells. After electrophoresis and transfer, the PVDF membranes were incubated and shake overnight with primary antibodies at 4°C. Then, the imprints were washed with TBST for 3 times and cultured with incubated with an HRP-conjugated secondaey antibody (diluted at 1:5000) at room temperature for 1 hour. The proteins were visualized using a Immobilon^TM Western Chemiluminescent HRP substrate (Millipore, USA). The primary antibodies used were: Alpha Tubulin (Proteintech, 66031-1-lg), BCL2 (Proteintech,60178-1-lg), HIF-1α (Proteintech, 20962-1-AP), MMP9 (Proteintech, 10375-2-AP), PIM1 (Cell Signaling Technology, 3247T), STAT3 (Cell Signaling Technology, 9139), p-STAT3 (Cell Signaling Technology, 9145), TRIM44 (Proteintech, 11511-1-AP) and SPATS2 (Santa Crus, sc-390306).

Proteins of GCs were obtained by RIPA lysis buffer RIPA (Cat. No. P0013B, Beyotime) supplemented with a protease inhibitor cocktail in centrifuged tubes in ice for 20 min. The concentration of the protein was measured by using the BCA protein assay (Cat. No. 23225, Thermo Fisher Scientific). 30 μg of proteins from each sample were loaded to SDS-PAGE before transferring to nitrocellulose membranes (Bio-Rad Biotechnology, Hercules, CA, USA), and probed with the indicated antibodies (CASPASE 9: Cat. No. 10380-1-AP, Proteintech, 1:1000; BAX: Cat. No. 50599-2-Ig, Proteintech, 1:1000; BCL 2: Cat. No. 26593-1-AP, Proteintech, 1:1000; PCNA: Cat. No. 10205-2-AP, Proteintech, 1:1000; ALPHA TUBULIN: Cat. No. 11224-1-AP, Proteintech, 1:1000). Next day, the peroxidase-conjugated secondary antibody (Cat. No. SA00001-2, Proteintech, 1:2000) was added to incubate the mem-brane. Bands were visualized with the Clarity™ Western ECL Substrate (Cat. No. 32209, Thermo Fisher Scientific) and quantified with Image J. All samples were performed in triplicate.