Ki67 ab15580

Primary antibodies against OGT (24083) and O-GlcNAc (9875) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β-actin (A300–491A) were purchased from Bethyl Laboratories (Montgomery, TX, USA), and those against PRKCG (SC-166451) and PDGFRB (SC-374573) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies against horseradish peroxidase-linked anti-rabbit (A120–101P) and anti-mouse (A90–116P) antibodies were purchased from Bethyl Laboratories. For immunohistochemistry (IHC), primary antibodies against OGT (ab96718) and Ki67 (ab15580) were purchased from Abcam (Cambridge, UK), and antibodies against Caspase-3 (9661) were purchased from Cell Signaling Technology. GEM (G6423) was purchased from Selleck Chemicals (Houston, TX, USA). PTX (T7402), Thiamet G (SML0244), MG132 (M7449), and phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma-Aldrich. MTT (M1415) was purchased from Duchefa Biochemie (Haarlem, The Netherlands). Protease-inhibitor cocktail tablets were purchased from Roche Applied Biosciences, and RNAi-Max (13778150) was purchased from Thermo Fisher Scientific.

The cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature, washed with PBS for 3 times, and incubated with 0.5% Triton X-100 for 20 min. The proliferation status of the cells was then measured using BeyoClick™ EdU Cell Proliferation Kit (C0071, Beyotime, Shanghai). The tumor samples were collected, incubated in 4% PFA for fixation for 12 h, and sectioned into 20 μm slices. The slices were further incubated in 1% Triton X-100, primary antibody solution (Ki67, ab15580, Abcam, American) and secondary antibody solutions in sequence before imaging.

Antibodies for Taz (sc-48,805), Gli1 (sc-20,687), Gli2 (sc-271,786), Gli3 (sc-74,478), c-myc (sc-40), PKAc (sc-903), p-PKA (T198, sc-32,968), GSK3β (sc-9166), CK1α (sc-74,582), acetylated-α-tubulin (sc-23,950), β-actin (sc-69,879), Lamin B (sc-374,015), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-32,233), and rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). YAP (#12395), p-GSK3β (Ser9/21, #8566), Lats1 (#9153), HA (#3724), and Flag (#14793) were from Cell Signaling Technology (Danvers, MA).Gli3 (ab69838, immunogen corresponding to the residues 1–100 of human Gli3), Gli3 (ab181130, immunogen corresponding to the residues 1300–1500 of human Gli3), Smo (ab72130), p-Ser/Thr (ab117253) and Ki67 (ab15580) were purchased from Abcam (Cambridge, UK). Ptc1 (06–1102) was from Millipore (Billerica, MA). The IRDye 680 and 800 second antibodies were from LI-COR Bioscience (Lincoln, NE). GST fusion proteins, including GST-Gli3F, GST-Gli3R, and GST-Taz, were generated as previously described (Einarson et al., 2007). Bioactive Shh recombinant protein (N-Shh) was from PeproTech Inc. (Rocky Hill, NJ), whereas H-89, SB216763, and forskolin were from Sigma (St. Louis, MO).

Extraction of total proteins, Western blot, and immunoprecipitations analyses were performed as described (Sonego et al, 2013; Fabris et al, 2015; Lovisa et al, 2016). Primary antibodies used were as follows: vinculin (N‐19, 1:1,000), CDK6 (C‐21, sc‐177, 1:800), CDK4 (C‐22, sc‐260, 1:400), pRB1S780 (sc‐12901, 1:400), PSF (39‐1, sc‐101137, 1:800), CHK1 (G‐4, sc‐8408, 1:500), luciferase (sc‐32896), lamin A (C‐20 sc‐6214) (Santa Cruz Biotechnology); FOXO3 (75D8, #2497, 1:600), cyclin D3 (DCS22, #2936, 1:500), pCHK1S296 (#2349, 1:500), H2AX (D17A3, #7631, 1:500), ATR (#2790, 1:500), ATM (#2873, 1:500), and cleaved caspase‐3 Asp175 (#9661, 1:500) (Cell Signaling); cyclin D1 (DCS‐6, CC12, 1:1,000) (Millipore); γH2AXS139 (#2577, 1:500) (Upstate Biotechnology); RB1 (554136, 1:500) and GRB2 (610111, 1:300) (BD Biosciences); actin (A5060, 1:500), tubulin (T5168, 1:1,000), Flag (F3040), OP18 (O0138, 1:1,000), and V5 (A7345)‐ and HA (A2095)‐agarose conjugated (Sigma‐Aldrich Co); GFP (11 814 440 001, 1:500) (Roche); Ki67 (ab15580, 1:1,000) (Abcam). Quantification of the blots was done using the QuantiONE software (Bio‐Rad Laboratories) or the Odyssey infrared imaging system (LI‐COR Biosciences).

Immunohistochemistry was used to detect the expression of UCHL1 in lung adenocarcinoma. The slices were placed in an oven at 60 °C for 20 min. After removal, the slices were soaked in xylene solution twice for 10 min and then soaked in 100%, 95%, 90%, and 80% ethanol for 5 min. After cleaning with PBS solution, the slices were incubated in 3% hydrogen peroxide for 20 min and then washed with PBS solution. After drying, the normal goat serum was dripped into the slice surface and incubated at 37 °C for 30 min. The primary antibody (UCHL1/#13179 cell signaling technology, Ki-67/ab15580 abcam) was taken out and added into the wet box at 4 °C overnight. After washing with PBS, the labeled second antibody was added and incubated at 37 °C for 30 min. Then, PBS was washed again and added with a chromogenic agent for routine re-staining, dehydration, and transparency. Finally, neutral gum was used to seal the slices, and the color development intensity was observed under the microscope. The following are the immunohistochemical staining intensity criteria: negative (0–1), weak positive (1–2), medium (2–3), and strong positive (≥ 3).