Skip to main content
. 2017 Mar 31;6:e23172. doi: 10.7554/eLife.23172

Figure 1. Ambra1 interacts with FAK at focal adhesions.

FAK or Ambra1 were immunoprecipitated from FAK-WT and FAK -/- cell lysates using (A) anti-FAK 4.47 agarose or (B) anti-Ambra1, followed by western blot analysis with anti-FAK and anti-Ambra1. (C) FAK was immunoprecipitated from Ambra1 +/+ and Ambra1 -/- MEF cell lysates using anti-FAK 4.47 agarose, followed by western blot analysis with anti-FAK and anti-Ambra1. Anti-β-actin was used as a loading control. Relative ratios of Ambra1, FAK/Ambra1 and Ambra1/FAK interactions were calculated by densitometry. (D) FAK-WT and FAK -/- cells were seeded onto glass coverslips, fixed and stained using anti-Ambra1 and anti-Paxillin. Scale bars, 20 μm. (E, F) Focal adhesions were isolated from FAK-WT and FAK -/- cells using hydrodynamic force. (E) Focal adhesions (solid arrows) were stained with anti-FAK and anti-Ambra1 (left panels) and with anti-Ambra1 and anti-Paxillin (right panels). (F) Focal adhesions (solid arrows) were stained with anti-Ambra1 and anti-Rack1 in SCC FAK-WT (left panels) and SCC FAK -/- cells (right panels). Scale bars, 20 μm. Colocalisation (Costes r value from five cells) was analysed using the ImageJ plugin JaCoP (Bolte and Cordelières, 2006).

DOI: http://dx.doi.org/10.7554/eLife.23172.003

Figure 1—source data 1. COSTES r values for immunofluorescence images.
COSTES mean and s.d. values for Figure 1D–F are shown.
DOI: http://dx.doi.org/10.7554/eLife.23172.004
elife-23172-fig1-data1.xlsx (44.2KB, xlsx)
DOI: 10.7554/eLife.23172.004

Figure 1.

Figure 1—figure supplement 1. Ambra1 +/+ and -/- mouse embryonic fibroblasts (MEFs).

Figure 1—figure supplement 1.

(A) Representative images of Ambra1 +/+ and Ambra1 -/- MEFs. (B) PCR of Ambra1 +/+ and Ambra1 -/- MEFs. B2M served as a control for equal input. (C) SCC FAK-WT and FAK -/- cells were grown on glass coverslips for 24 hr, fixed and stained with anti-Ambra1, anti-CoxIV and DAPI. (D, E) Focal adhesions were isolated from FAK-WT and FAK -/- cells using hydrodynamic force. Focal adhesions (solid arrows) were stained with anti-Ambra1 and anti-CoxIV (D) or anti-FAK and anti-CoxIV (E) in SCC FAK-WT (left panels) and SCC FAK -/- cells (right panels). Scale bars, 20 μm. Colocalisation (Costes r value from five cells) was analysed using the ImageJ plugin JaCoP. (F, G) Total Internal Reflection Fluorescence (TIRF) microscopy of SCC FAK-WT and -/- cells stained with anti-Ambra1 and anti-FAK (F) or anti-Ambra1 and anti-pSrc Y416. (G) Colocalisation (COSTES r value of five cells) was analysed using the ImageJ plugin JaCoP. Scale bars, 10 μm.
DOI: http://dx.doi.org/10.7554/eLife.23172.005
Figure 1—figure supplement 2. Knockdown of Ambra1 suppresses FAK phenotypes.

Figure 1—figure supplement 2.

(A) Polarity assay: FAK-WT and FAK -/- cells were transiently transfected with either a pool or two independent Ambra1 siRNAs. A confluent monolayer of cells plated on fibronectin was wounded using a pipette tip, fixed 1.5 hr later and stained with anti-GM130 (Golgi), TRITC-phalloidin and DAPI. The orientation of the Golgi towards to wound edge was used to score polarisation. Scale bars, 20 μm. (B) Quantification of the polarity assay in SCC FAK-WT and -/- cells. n = 3. Error bars, s.d. p<0.01. (C) Polarity assay in Ambra1 +/+ and -/- MEFs. n = 3. Error bars, s.d. p<0.01. (D, E) Invasion assay: Cells were seeded on growth factor-reduced Matrigel in serum-free conditions. After 72 hr invasion towards a serum gradient, cells were visualised by staining with calcein. (D) Quantification of the invasion assay. n = 5. Error bars, s.e.m. p<0.01 (*) and p<0.05 (#). (E) Representative images of the invasion assay.
DOI: http://dx.doi.org/10.7554/eLife.23172.006