Protein extraction and western blotting were performed as described elsewhere.58 (link) Antibodies directed against HMGA1 proteins were already described.59 (link) Commercial antibodies were: anti-pSer473-AKT (#9271), anti-AKT (#9272), anti-Caspase-7 (#9492), anti-phospho S6 ribosomal protein (Ser240/244) (D68F8, #5364), anti-S6 ribosomal protein (54D2, #2317S), anti-LC3A/B (#4108), anti-PARP (46D11, #9532), anti-ATG5 (D5F5U, #12994) and anti-ULK1 (R600, #4773) antibodies from Cell Signaling Technology (MA, USA); anti-β-Actin (I-19, sc-1616), anti-β-Tubulin (D-10, sc-5274), anti-p21 (C-19, sc-397), anti-Cyclin D1 (A12, sc-8396), anti-Cyclin E (E20, sc-481), and anti-Vinculin (N-19, sc-7649) were from Santa Cruz Biotechnology (Dallas, TX, USA); anti-p27 (610241) and anti-p62/SQSTM1 (610833) were from BD Biosciences (Franklin Lakes, NJ USA); anti-phospho ATG13 (Ser318) (PAB19948) was from Abnova (Taipei City,Taiwan); anti-ATG13 (SAB4200100) was from Sigma-Aldrich. ECL System was purchased from Amersham Pharmacia (Buckinghamshire, UK).
Protocol detail
Protein Extraction and Western Blotting
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Corresponding Organization :
Other organizations : Institute for Experimental Endocrinology and Oncology, University of Naples Federico II, Addenbrooke's Hospital, University of Cambridge, Wellcome Trust
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Variable analysis
independent variables
- Protein extraction and western blotting procedures
- Expression and phosphorylation levels of proteins (HMGA1, pSer473-AKT, AKT, Caspase-7, phospho S6 ribosomal protein, S6 ribosomal protein, LC3A/B, PARP, ATG5, ULK1, β-Actin, β-Tubulin, p21, Cyclin D1, Cyclin E, Vinculin, p27, p62/SQSTM1, phospho ATG13)
- Antibodies used for western blotting
- ECL System for western blotting
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