Journal: F1000Research

Article Title: Murine Tim-1 is excluded from the immunological synapse

doi: 10.12688/f1000research.1-10.v2

Figure Lengend Snippet: ( A ) Jurkat T cells transfected with empty vector (EV), Flag-Tim-1, or Flag-Tim-1 QGQ were stimulated with anti-TCR and anti-CD28 antibodies for the indicated times. Lysates were analyzed by SDS-PAGE and western blotting for pY (4G10), pSrc (Y416; analogous to Y394 in Lck), and β-actin. ( B ) Jurkat T cells were stimulated with anti-CD3 mAb for the indicated times. CD3 expression was measured with flow cytometry and mean fluorescence intensity (MFI) was determined in FloJo. Dynasore (DS, 80 µM) was used to prevent clathrin-mediated endocytosis after TCR/CD3 crosslinking.

Article Snippet: The following antibodies were used: pSrc Y416 and pZAP-70 Y319 (Cell Signaling), PKC-θ (C-18, Santa Cruz), CD43 (clone S7, BD Pharmingen), M2-Cy3 (Sigma Aldrich), EEA1 (BD Transduction), M2 anti-flag (Sigma Aldrich), anti-human CD3 (Becton Dickinson), mouse CD3 and CD28 (BD Pharmingen), human CD28 (Life Technologies), Tim-1 Fc (eBiosciences), anti-TCR antibody C305 (Harlan), anti-Tim-1 antibodies (3B3 and 5F12), anti-Tim-4 antibodies (3A1, 3H11 and 5G3).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Expressing, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms

doi: 10.1371/journal.pone.0004686

Figure Lengend Snippet: A, Effect of siRNA depletion of c-Src on ghrelin-induced Akt phosphorylation. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM) at 37°C. After stimulation, equal amounts of protein in each sample were used to assess the expression of c-Src (left panel) and Akt phosphorylation (right panel) by western blotting. Expression of c-Src was quantified by densitometry and expressed as percentages of the level of c-Src in control siRNA-transfected cells (mean±S.E.). Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) after ghrelin addition to control siRNA-transfected cells (mean±S.E.). B, Effect of ghrelin on Y phosphorylation of Akt and interaction between Akt and c-Src. Cells were incubated with ghrelin (100 nM, 5 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with pAkt HM(S473), pY, pSrc (Y416) antibodies. C, Effect of ghrelin on Y phosphorylation of Akt in the presence of c-Src siRNA. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM, 5 min) at 37°C. Equal amounts of protein in each sample were used to assess the expression of c-Src [upper panel; values shown (mean±S.E.) are percentages of the level of c-Src in control siRNA-transfected cells]. Cells were lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with with pAkt HM(S473), pY, pSrc (Y416) antibodies. Immunoblots are representative of three independent experiments.

Article Snippet: Anti-p44/42 MAPK rabbit polyclonal, anti-pSrc (Y416) rabbit polyclonal, anti-pAkt HM (S473), anti-pAkt A-loop (T308), anti-Akt rabbit polyclonal, anti-Rictor rabbit polyclonal, anti-mTOR rabbit polyclonal and anti-pPDK1 (S241) rabbit polyclonal antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Transfection, Expressing, Western Blot, Control, Incubation, Immunoprecipitation

Journal: PLoS ONE

Article Title: c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms

doi: 10.1371/journal.pone.0004686

Figure Lengend Snippet: A, Effects of the Src inhibitor PP2 and its negative control, PP3, on the ghrelin-induced Akt activation in 3T3-L1 preadipocyte cells. Serum-starved cells were pretreated with PP2 (5 µM, 30 min) or PP3 (5 µM, 30 min) before ghrelin stimulation (100 nM) for the indicated time periods. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) (mean±S.E of three independent experiments). B, Effect of ghrelin on the assembly of complexes containing β-arrestins 1 and 2 and pAkt. Preadipocyte 3T3-L1 cells were incubated with ghrelin (100 nM, 10 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to β-arrestins 1 and 2, and then analyzed by western blotting with pAkt HM (S473), pAkt A-loop (T308), pY, pSrc (Y416) antibodies. For A and B, western blots are representative of three independent experiments.

Article Snippet: Anti-p44/42 MAPK rabbit polyclonal, anti-pSrc (Y416) rabbit polyclonal, anti-pAkt HM (S473), anti-pAkt A-loop (T308), anti-Akt rabbit polyclonal, anti-Rictor rabbit polyclonal, anti-mTOR rabbit polyclonal and anti-pPDK1 (S241) rabbit polyclonal antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Negative Control, Activation Assay, Phospho-proteomics, Incubation, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( 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

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Control, Staining, Isolation, Fluorescence, Microscopy

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( A, B ) SCC FAK-WT, FAK P875A/P881A (AA) and FAK -/- cells were grown on glass coverslips, fixed and stained with anti-FAK, anti-Paxillin and DAPI ( A ) and anti-Ambra1, anti-Paxillin and DAPI ( B ), respectively. Scale bars, 20 μm. There were no visible differences between total FAK or Ambra1 intensity staining. ( C, D ) Total Internal Reflection Fluorescence (TIRF) microscopy of SCC FAK P875A/P881A cells stained with anti-Ambra1 and anti-FAK ( C ) or anti-Ambra1 and anti-pSrc Y416 ( D ). Colocalisation (COSTES r value of five cells) was analysed using the ImageJ plugin JaCoP. Scale bars, 10 μm. ( E ) FAK was immunoprecipitated from FAK-WT, FAK P875A/P881A (AA) and FAK -/- cell lysates using anti-FAK 4.47 agarose, followed by western blot analysis with anti-FAK and anti-Src. The relative ratio of Src/FAK interaction was calculated by densitometry. DOI: http://dx.doi.org/10.7554/eLife.23172.018

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Staining, Fluorescence, Microscopy, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( A, B ) Src ( A ) or pSrc Y416 ( B ) were immunoprecipitated from FAK-WT and FAK -/- cell lysates using anti-Src agarose or anti-pSrc Y416 antibody, followed by western blot analysis with anti-Ambra1, anti-pSrc Y416 and anti-Src. Relative ratios of Ambra1/Src and Ambra1/pSrc interactions were calculated by densitometry. ( C ) FAK-WT and FAK -/- cells were seeded onto glass coverslips, fixed and stained using anti-pSrc Y416, anti-Ambra1 and DAPI. Scale bars, 20 μm. ( D ) SCC FAK-WT and FAK -/- cells were grown on glass coverslips, fixed and stained with anti-Ambra1, anti-LC3B and DAPI. Scale bars, 20 μm. ( E ) LC3B was immunoprecipitated from SCC FAK-WT and FAK -/- cell lysates using anti-LC3B, followed by western blot analysis with anti-Ambra1 and anti-LC3B. Relative ratios of LC3B II/LC3B I as well as the Ambra1/LC3B and Ambra1/LC3B II interactions were calculated by densitometry. ( F – J ) SCC FAK-WT and FAK -/- cells were transiently transfected with either a pool ( F – H ) or two individual siAmbra1 siRNAs ( F, I, J ). The cells were grown on glass coverslips, fixed and stained with anti-pSrc Y416, anti-Paxillin and DAPI. ( G, I ) Representative immunofluorescence images. Scale bars, 20 μm. ( H, J ) Quantification of internalised active Src. n = 3. Error bars, s.d. p<0.001. Colocalisation (Costes r value from five cells) was analysed using the ImageJ plugin JaCoP. DOI: http://dx.doi.org/10.7554/eLife.23172.007 10.7554/eLife.23172.008 Figure 2—source data 1. COSTES r values for immunofluorescence images and percentage of cells with internalised pSrc. COSTES mean and s.d. values for are shown. Mean percentage and s.d. values of cells with internalised pSrc upon transient Ambra1 knockdown by siRNA in SCC FAK-WT and -/- cells are shown . DOI: http://dx.doi.org/10.7554/eLife.23172.008

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Staining, Transfection, Immunofluorescence, Knockdown

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( A ) Nuclei from SCC FAK-WT and FAK -/- cells were isolated using sucrose gradient centrifugation. Lysates were immunoblotted for Ambra1, GAPDH (cytosolic marker) and Lamin A/C (nuclear marker). ( B ) Relative ratio of Ambra1/Lamin A/C was calculated by densitometry. WCL, whole cell lysate. Error bars, s.d. ( C ) Colocalisation (Costes r value from five cells) of pSrc Y416/Paxillin upon Ambra1 knockdown by siRNAs was analysed using the ImageJ plugin JaCoP. Error bars, s.d. p<0.01 (*) and p<0.05 (#). ( D ) FAK-WT and FAK -/- cells were transiently transfected with either a pool or two independent Ambra1 siRNAs. Cell lysates were subjected to western blot analysis using anti-Ambra1, anti-pSrc Y416 and anti-Src. Anti-GAPDH was used as a loading control. ( E ) The relative ratios of pSrc/GAPDH were calculated using densitometry. n = 3. Error bars, s.d. p<0.01 (*) and p<0.05 (#). DOI: http://dx.doi.org/10.7554/eLife.23172.009

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Isolation, Gradient Centrifugation, Marker, Knockdown, Transfection, Western Blot, Control

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( A ) Network analysis of Ambra1-, FAK- and pSrc Y416-interacting proteins that are involved in trafficking processes. Solid lines indicate protein-protein interactions identified in the mass spectrometry datasets used for the interaction map. Dotted lines indicate Ambra1–FAK/pSrc interactions, which have been previously identified and verified by immunoprecipitation. ( B ) Ambra1 was immunoprecipitated from FAK-WT and FAK -/- cell lysates using anti-Ambra1 antibody, followed by western blot analysis with anti-Dctn1 and anti-Ambra1. ( C – E ) Ambra1 ( C ), FAK ( D ) or pSrc Y416 ( E ) were immunoprecipitated from FAK-WT and -/- cell lysates, followed by western blot analysis with anti-IFITM3, anti-Ambra1, anti-FAK and anti-pSrc Y416. Anti-GAPDH served as a loading control. Relative ratios of Dctn1/Ambra1, IFITM3/Ambra1, IFITM3 and IFITM3/pSrc interactions were calculated by densitometry. DOI: http://dx.doi.org/10.7554/eLife.23172.010

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Protein-Protein interactions, Mass Spectrometry, Immunoprecipitation, Western Blot, Control

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( A ) Focal adhesions from SCC FAK-WT and FAK -/- cells were crosslinked and isolated for western blot analysis with the indicated antibodies. Paxillin served as a marker for focal adhesions, Lamin A/C was used as a nuclear marker and GAPDH represented a cytosolic marker. WCL, whole cell lysate; FA, focal adhesions. SCC FAK-WT and -/- cells were transiently transfected with siIFITM3 and pSrc Y416 ( B ) and FAK ( D ) were immunoprecipitated from FAK-WT and FAK -/- cell lysates using anti-pSrc Y416 and anti-FAK 4.47 agarose, followed by western blot analysis with anti-Ambra1, anti-FAK, anti-IFITM3, anti-pSrc Y416 and anti-Src. Anti-GAPDH served as a loading control. ( C, E ) Relative ratios of Ambra1/pSrc ( C ) as well as pSrc/FAK and Src/FAK ( E ) interactions were calculated by densitometry. n = 3. Error bars, s.d. p<0.05 (#). DOI: http://dx.doi.org/10.7554/eLife.23172.011

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Isolation, Western Blot, Marker, Transfection, Immunoprecipitation, Control

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( A ) FAK-WT and FAK -/- cells were transiently transfected with a pool of Dynactin 1 (Dctn1) siRNAs and lysed 48 hr post transfection. Dynactin 1 expression was determined by western blotting using anti-Dctn1. Anti-GAPDH was used as a loading control. ( B ) SCC FAK-WT and FAK -/- cells transiently transfected with Dctn1 siRNA were grown on glass coverslips, fixed and stained with anti-pSrc Y416, anti-Paxillin and DAPI. Scale bars, 20 μm. ( C ) Quantification of internalised active Src. n = 3. Error bars, s.d. p<0.01. ( D ) FAK-WT and FAK -/- cells were transiently transfected with a pool of IFITM3 siRNAs and lysed 48 hr post transfection. IFITM3 expression was determined by western blotting using anti-IFITM3. Anti-GAPDH was used as a loading control. ( E ) SCC FAK-WT and FAK -/- cells transiently transfected with IFITM3 siRNA were grown on glass coverslips, fixed and stained with anti-pSrc Y416, anti-Paxillin and DAPI. Scale bars, 20 μm. ( F ) Quantification of internalised active Src. n = 3. Error bars, s.d. p<0.01. DOI: http://dx.doi.org/10.7554/eLife.23172.012 10.7554/eLife.23172.013 Figure 4—source data 1. Percentage of cells with internalised pSrc. Mean percentage and s.d. values of cells with internalised pSrc upon transient Dctn1 or IFITM3 knockdown by siRNA in SCC FAK-WT and -/- cells are shown. DOI: http://dx.doi.org/10.7554/eLife.23172.013

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Transfection, Expressing, Western Blot, Control, Staining, Knockdown

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: SCC FAK-WT and FAK -/- cells transiently transfected with siDctn1 ( A ) or siIFITM3 ( B ) were grown on glass coverslips, fixed and stained with anti-pSrc Y416, anti-Paxillin and DAPI. Colocalisation (Costes r value from nine or five cells respectively) of pSrc/Paxillin was analysed using the ImageJ plugin JaCoP. Error bars, s.d. p<0.01 (*) and p<0.05 (#). DOI: http://dx.doi.org/10.7554/eLife.23172.014

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Transfection, Staining

Journal: eLife

Article Title: Ambra1 spatially regulates Src activity and Src/FAK-mediated cancer cell invasion via trafficking networks

doi: 10.7554/eLife.23172

Figure Lengend Snippet: ( A, B ) FAK ( A ) or Ambra1 ( B ) were immunoprecipitated from FAK-WT, FAK P875A/P881A (AA) and FAK -/- cell lysates using anti-FAK 4.47 agarose or anti-Ambra1, followed by western blot analysis with anti-FAK, anti-Ambra1 and anti-p130Cas. Anti-GAPDH was used as a loading control. ( C ) Relative ratios of Ambra1/FAK and p130Cas/FAK interactions were calculated by densitometry. ( D ) Adhesion assay: SCC FAK-WT, FAK P875A/P881A and FAK -/- cells were plated in serum-free conditions on fibronectin-coated plates. Samples were normalised to the 6 hr time point and relative adhesion was calculated by setting the FAK-WT values to 1. n = 3. Error bars, s.d. p≤0.01. ( E, F ) SCC FAK-WT, FAK P875A/P881A and FAK -/- cells were grown on glass coverslips for 24 hr, fixed and stained with anti-pSrc Y416, anti-Paxillin and DAPI. Scale bars, 20 μm. The relative intensity of pSrc staining at focal adhesions from five cells (at least 10 focal adhesions/cell) was measured using ImageJ. ( E ) Representative immunofluorescence images are shown. ( F ) Quantification of the relative pSrc intensity at focal adhesions. n = 5. Error bars, s.e.m. p<0.01 (*) and p<0.05 (#). ( G ) Focal adhesions from SCC FAK-WT, FAK P875A/P881A (AA) and FAK -/- cells were crosslinked and isolated for western blot analysis with the indicated antibodies. Paxillin and Talin served as markers for focal adhesions and Lamin A/C was used as a nuclear marker. Hsp90 and GAPDH represented cytosolic markers. WCL, whole cell lysate; FA, focal adhesions. The purity of the isolated focal adhesions was determined by the absence of nuclear proteins like Lamin A/C and cytosolic markers like Hsp90 and GAPDH. There was less active Src at focal adhesions in the FAK-deficient SCC cells due to Src’s internalisation to autophagic structures. Additionally, increased pPaxillin Y118 and Talin levels could be detected in the FAK Ambra1-binding mutant compared to SCC FAK-WT and FAK -/- cells. No changes in Ambra1 levels at focal adhesions could be detected. ( H ) The relative ratios of pFAK/FAK, pSrc/Src and Ambra1 at focal adhesions were calculated using densitometry. n = 3. Error bars, s.d. p<0.01 (*) and p<0.05 (#). DOI: http://dx.doi.org/10.7554/eLife.23172.015 10.7554/eLife.23172.016 Figure 5—source data 1. Relative mean values of adhesion, pSrc intensity at focal adhesions and relative ratios at focal adhesions. Mean and s.d. values of relative adhesion on fibronectin of SCC FAK-WT, P875A/P881A and -/- cells are shown . The relative mean intensity and s.e.m. of pSrc at focal adhesions are shown . Relative ratios (mean and s.d.) of pFAK/FAK, pSrc/Src and Ambra1 of isolated focal adhesions are shown . DOI: http://dx.doi.org/10.7554/eLife.23172.016

Article Snippet: Antibodies used were as follows: anti-Paxillin (RRID: AB_647289 ), anti-GM130 (RRID: AB_398141 ), anti-p130Cas (RRID: AB_397667 ) and anti-RACK1 (RRID: AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID: AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID: AB_10694213 ), anti-FAK (RRID: AB_10694068 ), anti-pFAK Y397 (RRID: AB_2173659 ), anti-pPaxillin Y118 (RRID: AB_2174466 ), anti-Rab7 (RRID: AB_1904103 ), anti-pSrc Y416 (RRID: AB_331697 ), anti-Src (clone 36D10) (RRID: AB_10693939 ), anti-LC3B (RRID: AB_2137707 ) and anti-GAPDH (RRID: AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID: AB_10842517 ), anti-Ambra1 (RRID: AB_2636939 ) and anti-pSrc Y416 (RRID: AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID: AB_310789 ) (Millipore, Billerica, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Control, Cell Adhesion Assay, Staining, Immunofluorescence, Isolation, Marker, Binding Assay, Mutagenesis