Western Blotting and Cell Fractionation

Western blotting was performed as already described^{55 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643), mouse anti-cyclin A2 (#4656), rabbit anti-BCL2 (#2876), rabbit anti-BAX (#2772), rabbit anti-cyclin B1 (#12231), rabbit anti-PARP-1 (#9532), rabbit anti-phospho-ATR (Ser428) (#2853), rabbit anti-phospho-CHK1 (Ser345) (#2348), rabbit anti-phospho-CDC2 (Tyr15) (#4539), rabbit anti-phospho-H2A.X (Ser139) (#9718), rabbit anti-phospho-Histone H3 (Ser10) (#3377), rabbit anti-phospho-WEE1 (Ser642) (#4910) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CDC2 (sc-954), mouse anti-Myc (sc-40), mouse anti-HSP90 (sc-13119) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-SMO (ST1718) (Merck Millipore, Burlington, MA, USA). Chemiluminescent detection was used. Cell fractionation was performed as previously described^{56 (link)}. The following antibodies were used: mouse anti-GLI1 (#2643) (Cell Signaling Technology), goat anti-fibrillarin (D-14), and goat anti-GAPDH (V-18) (Santa Cruz Biotechnology).

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Pietrobono S., Santini R., Gagliardi S., Dapporto F., Colecchia D., Chiariello M., Leone C., Valoti M., Manetti F., Petricci E., Taddei M, & Stecca B. (2018). Targeted inhibition of Hedgehog-GLI signaling by novel acylguanidine derivatives inhibits melanoma cell growth by inducing replication stress and mitotic catastrophe. Cell Death & Disease, 9(2), 142.

Publication 2018

mouse anti-GLI1 mouse anti-cyclin A2 rabbit anti-BCL2 rabbit anti-BAX rabbit anti-cyclin B1 rabbit anti-PARP-1 rabbit anti-phospho-ATR (Ser428) rabbit anti-phospho-CHK1 (Ser345) rabbit anti-phospho-CDC2 (Tyr15) rabbit anti-phospho-H2A.X (Ser139) rabbit anti-phospho-Histone H3 (Ser10) goat anti-fibrillarin (D-14), goat anti-GAPDH (V-18)

Anti b1 Antibodies Bcl2 Cdc2 Cell fractionation Cyclin Cyclin a2 Fibrillarin Gapdh Goat H2a x Histone h3 Hsp90 Mouse Parp 1 Rabbit

Corresponding Organization : Azienda Ospedaliero-Universitaria Careggi

Other organizations : Tumour Institute of Tuscany, Istituto di Fisiologia Clinica, University of Siena

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Treatment with antibodies: mouse anti-GLI1, mouse anti-cyclin A2, rabbit anti-BCL2, rabbit anti-BAX, rabbit anti-cyclin B1, rabbit anti-PARP-1, rabbit anti-phospho-ATR (Ser428), rabbit anti-phospho-CHK1 (Ser345), rabbit anti-phospho-CDC2 (Tyr15), rabbit anti-phospho-H2A.X (Ser139), rabbit anti-phospho-Histone H3 (Ser10), rabbit anti-phospho-WEE1 (Ser642), rabbit anti-CDC2, mouse anti-Myc, mouse anti-HSP90, and rabbit anti-SMO

dependent variables

Protein expression levels measured by Western blotting

control variables

Western blotting protocol as already described in the referenced study (link)
Cell fractionation protocol as previously described in the referenced study (link)

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

Annotations

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