Journal: Frontiers in Cell and Developmental Biology
Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization
doi: 10.3389/fcell.2020.00733
Figure Lengend Snippet: Specific loss of FIP200 results in elevated phospo-ERK1/2 across the time course of fibronectin-induced cell spreading. (A) MCF10A wild-type and FIP200 KO cells were kept in suspension in EGF depleted growth media for 1 hour prior to plating on 10 μg/ml fibronectin for the indicated times. Time zero indicates cells in suspension. Western blot analysis was performed on lysates harvested at the indicated times. Immunoblotting was performed against the indicated proteins. (B) Quantitation of Ser473 Akt phosphorylation western blot integrated density values normalized to actin from wild-type and FIP200 KO cells. (C) Fold change in Akt Ser473 or ERK1 Thr202 and ERK2 Tyr204 phosphorylation from zero time point following normalization to actin from wild-type and FIP200 KO cells. (D) MCF10A wild-type and Atg5 KO cells were kept in suspension in EGF depleted growth media for 1 hour prior to plating on 10 μg/ml fibronectin for the indicated times. Time zero indicates cells in suspension. Western blot analysis was performed on lysates harvested at the indicated times. Immunoblotting was performed against the indicated proteins. (E) Quantitation of Ser473 Akt phosphorylation western blot integrated density values normalized to actin from wild-type and Atg5 KO cells. (F) Fold change in Akt Ser473 or ERK1 Thr202 and ERK2 Tyr204 phosphorylation from zero time point following normalization to actin from wild-type and Atg5 KO cells. Results represent at least 3 independent experiments and error bars are the SEM. * p < 0.05.
Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.
Techniques: Western Blot, Quantitation Assay
