Mab anti gapdh

The whole rabbit polyclonal antibodies (pAbs) and mouse monoclonal antibodies (mAbs) were listed below: pAb anti‐PIWIL1 (Abcam, Cambridge, UK), pAb anti‐CLOCK (CST, Boston), pAb anti‐BMAL1 (CST), pAb anti‐MYC‐tag (Santa Cruz, USA), pAb anti‐HA‐tag (Santa), mAb anti‐GAPDH (Abcam), mAb anti‐GSK3β (Zen Bioscience, Chengdu, China), pAb anti‐pGSK3β(S9) (CST), pAb anti‐AKT (CST), pAb anti‐pAKT(S473) (CST), pAb anti‐SRC (CST), pAb anti‐PER2 (CST), pAb anti‐CRY1 (CST).

Chicken pAb anti-GFP, mAb anti-GAPDH, and mAb anti-N-terminal ERC1a (ELKS-30) against residues 21–40 of mouse/human ERC1a (Abcam, Cambridge, UK); rabbit pAb anti-GFP (Thermo Fisher Scientific, Paisley, UK); rabbit pAbs anti-FLAG and anti-LL5β, mAbs anti-α-actinin, anti-tubulin, and anti-talin (Sigma-Aldrich); mAb anti-vinculin (Upstate, Lake Placid, NY, USA); rabbit pAb against the C-terminus of liprin-α1 (amino acids 818–1202)^{36 (link)} pAb rabbit anti-liprin-α1 (Protein Tech, Chicago, IL, USA); mAbs anti-paxillin and anti-GIT proteins (BD Bioscience, San Jose, CA, USA); mAb anti-LL5 (cl. IH12) from J. Sanes^{39 (link)}; mAb anti Src cl. 327 from S. Courtneidge^{40 (link)}. Alexa-Fluor–conjugated secondary antibodies (Thermo Fisher Scientific, Paisley, UK); horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Bioscience, Little Chalfont, UK). Lipophilic dye Nile Red (Sigma Aldrich, St. Louis, MO, USA).

For western blot detection of P53, P16, and P21, proteins were solubilized in 8 M Urea lysis buffer (8 M Urea, 50 mM Tris-HCl, 0.1 M β-Mercaptoethanol, 1 mM DTT, 100 μg/ml phenylmethanesulfonylfluoride). Proteins (15 μg) were separated by 8% (P53) or 14% (P21, P16) SDS-PAGE, blotted onto polyvinyl difluoride (PVDF) membrane (Millipore, Corporation, Billerica, MA, USA), and revealed using the anti-P53 DO-7 mAb (1/1000; Dako, Glustrup, Denmark), the anti-P21 EA10 mAb (0.5 μg/ml; Abcam ab16767, Cambridge, UK), or the anti-human-P16 mAb (1/1 000, BD Biosciences, Le Pont de Claix, France). Membranes were re probed using the anti-GAPDH mAb (1/2000; Abcam 9484, Cambridge, UK) or the anti-β-Tubulin TUB2.1 mAb (1/5 000, Sigma Aldrich, St Louis, MO) for loading/transfer controls. Membranes were then analysed using the Luminata crescendo western HRP substrate (Millipore Corporation, Billerica, MA, USA). Quantification of P53 was performed using the ImageJ software. Quantification of P21 and P16 was performed using the Fusion-Capt software (Vilber Lourmat, Eberhardzell, Germany). For detection of SHH, 1μg of concentrated culture supernatants were subjected to 10% SDS-PAGE, blotted, and revealed using the N-SHH 167Ab polyclonal antibody [26 (link)]. Quantification of N-SHH was performed using GeneGnome device and GeneTools software (Syngene, Synoptics Ltd, Cambridge, UK).