Western Blot Analysis of Murine Cerebella

Murine cerebella and cells were lysed in Tris–HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM and protease inhibitors, while nuclear extraction was performed as already described (Po et al., 2017 (link)).
Lysates were separated on 8% or 6% acrylamide gel and immunoblotted using standard procedures. The following antibodies were used: anti-GLI1 (L42B10, Cell Signaling), anti-CASPASE-3 (D3R6Y, Cell Signaling), anti-β-ARRESTIN1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-ACTIN I-19 (sc-1616; Santa Cruz Biotechnology), anti-E2F1 C-20 (sc-193; Santa Cruz Biotechnology), anti-E2F1 (acetyl K120/K125) (AP10555SU-N, Acris Antibodies); anti-ZIC1 (ab72694; Abcam); anti-PARP p85 Fragment (G7342; Promega), anti-SP1 1C6 (sc-420X; Santa Cruz Biotechnology), anti-PCNA (2586; Cell signaling), anti-H3 (ab 1971, Abcam), and anti-GAPDH (ab8245; Abcam). HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were used in combination with enhanced chemiluminescence (ECL Amersham).

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Abballe L., Alfano V., Antonacci C., Cefalo M.G., Cacchione A., Del Baldo G., Pezzullo M., Po A., Moretti M., Mastronuzzi A., De Smaele E., Ferretti E., Locatelli F, & Miele E. (2023). β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum. Frontiers in Cell and Developmental Biology, 11, 990711.

Publication 2023

anti-GLI1 (L42B10, anti-CASPASE-3 (D3R6Y, anti-ACTIN I-19 (sc-1616; anti-PARP p85 Fragment anti-SP1 1C6 (sc-420X; HRP-conjugated secondary antibodies

Acrylamide Actin Antibodies Caspase 3 Cells Cerebella Chemiluminescence Deoxycholic acid E2f1 Edta Gapdh Murine Nacl Pcna Promega Protease inhibitors Pyrophosphate Sodium Tris

Corresponding Organization : Sapienza University of Rome

Other organizations : Centre de Recherche en Cancérologie de Lyon, Inserm, Centre National de la Recherche Scientifique, Istituti di Ricovero e Cura a Carattere Scientifico

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Variable analysis

independent variables

Murine cerebella and cells were lysed in Tris–HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM and protease inhibitors

dependent variables

Protein expression levels of GLI1, CASPASE-3, β-ARRESTIN1, ACTIN, E2F1, E2F1 (acetyl K120/K125), ZIC1, PARP p85 Fragment, SP1, PCNA, H3, and GAPDH

control variables

Nuclear extraction was performed as already described (Po et al., 2017)

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