Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: HSP70 overexpression antagonizes cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with GFP-HSP70 plasmid or control plasmid. After 24 h, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) Nuclear morphology of apoptotic cells was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Apoptosis rate was determined by flow cytometry (representative plots and quantification is shown). ** P<0.01, with comparisons indicated by lines. HSP70, heat shock protein 70; GFP, green fluorescent protein; PARP, poly-ADP-ribose-polymerase; DDP, cisplatin; CON, control.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Staining, Expressing, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: Pretreatment with an HSP70 inhibitor enhances cisplatin-induced HGC-27 cells apoptosis. (A) HGC-27 cells were treated with different concentrations of PES for 24 h, and cell viability was measured using a Cell Counting Kit-8 assay. ** P<0.01 compared with untreated cells. (B) HGC-27 cells were pretreated with PES (8 µ M) for 2 h and then stimulated with cisplatin (5 µ g/ml) for 24 h. Expression levels of apoptosis-related proteins PARP, cleaved-caspase-3 and pro-caspase-3 were detected by western blotting. (C) Apoptosis rate was determined by flow cytometry (representative plots and quantification is shown). ** P<0.01 with comparisons indicated by lines. HSP70, heat shock protein 70; PES, pifithrin- µ ; PARP, poly-ADP-ribose-polymerase; DDP, cisplatin.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Cell Counting, Expressing, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: HSP70 downregulation enhances cisplatin-induced HGC-27 cell apoptosis. HGC-27 cells were transfected with HSP70 shRNA plasmid and control plasmid, and at 48 h post-transfection, cells were stimulated with 5 µ g/ml cisplatin for the indicated times. (A) The morphology of apoptotic cell nuclei was detected by DAPI staining (magnification, ×100). (B) Expression levels of apoptosis-related proteins PARP, pro-caspase-3 and cleaved caspase-3 were detected by western blotting. (C) Flow cytometry was used to determine apoptosis rates (representative plots and quantification is shown). ** P<0.01 with comparisons indicated by lines. HSP70, heat shock protein 70; sh, short hairpin; PARP, poly-ADP-ribose-polymerase; DDP, cisplatin; CON, control.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Transfection, shRNA, Plasmid Preparation, Staining, Expressing, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: HSP70 affects cisplatin-induced MAPK signaling pathway activation. (A) HGC-27 cells were treated with cisplatin (5 µ g/ml) for different amounts of time and the phosphorylation and total protein levels of (A) p38, ERK and JNK, and (B) Src, Akt and IκB, were monitored by western blotting. (C) HSP70-overexpressing and control plasmids were transfected into HGC-27 cells. (D) HGC-27 cells were pretreated with PES for 2 h and then stimulated with cisplatin for 6 or 8 h. (E) HSP70-shRNA and control plasmids were transfected into HGC-27 cells. After transfection, the cells were treated with cisplatin for 6 or 8 h and the phosphorylation and total protein levels of p38, ERK and JNK were monitored by western blotting. * P<0.05 compared with control cells at 6 h; # P<0.05 compared with control cells at 8 h. HSP70, heat shock protein 70; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; Src, SRC proto-oncogene non-receptor tyrosine kinase; Akt, AKT serine/threonine kinase 1; IκB, inhibitor of κB; sh, short hairpin; DDP, cisplatin; CON, control; p-, phosphorylated; PES, pifithrin- µ .

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Transfection, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: MAPK pathway inhibition enhances cisplatin-induced HGC-27 cell apoptosis. (A) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 24 h. Expression levels of PARP, cleaved caspase-3 and pro-caspase-3 were detected by western blotting. (B) Apoptotic rate was determined by flow cytometry (representative plots and quantification is shown). (C) HGC-27 cells were pretreated with specific inhibitors for p38, ERK or JNK for 2 h and then treated with cisplatin for 6 h. Phosphorylation of p38, ERK, JNK and the levels of HSP70 were detected by western blotting. * P<0.05 and ** P<0.01, with comparisons indicated by lines. MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PARP, poly-ADP-ribose-polymerase; HSP70, heat shock protein 70; DDP, cisplatin; p-, phosphorylated.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Inhibition, Expressing, Western Blot, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Inducible HSP70 antagonizes cisplatin-induced cell apoptosis through inhibition of the MAPK signaling pathway in HGC-27 cells

doi: 10.3892/ijmm.2018.3789

Figure Lengend Snippet: Schematic diagram illustrating the signaling pathway involved in the regulation of HSP70 in cisplatin-induced HGC-27 cell apoptosis. HSP70, heat shock protein 70; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: Rabbit monoclonal antibodies against p38 (cat. no. 9219s; 1:1,000), phosphorylated (p)-p38 (Thr180/Tyr182; cat. no. 9215s; 1:1,000), extracellular signal-regulated kinase (ERK, cat. no. 9102s; 1:1,000), p-ERK (Thr202/Tyr204; cat. no. 4376s; 1:1,000), c-Jun N-terminal kinase (JNK; cat. no. 9252s; 1:1,000), p-JNK (Thr183/Tyr185; cat. no. 4671s; 1:1,000), p-SRC proto-oncogene non-receptor tyrosine kinase (Src, Tyr416; cat. no. 6943s; 1:500), p-AKT serine/threonine kinase 1 (Akt, Ser473; cat. no. 4060s; 1:500), p-inhibitor of κB (IκB, Ser32; cat. no. 2859s; 1:500), poly-ADP-ribose-polymerase (PARP, cat. no. 9532s; 1:1,000), pro-caspase-3 (cat. no. 9662s; 1:500), cleaved caspase-3 (cat. no. 9661s; 1:500), β-actin (cat. no. 4970s; 1:1,000), GAPDH (cat. no. 5174s; 1:1,000), and rat monoclonal antibody against HSP70 (cat. no. 4573s; 1:1,000) were all from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques:

Journal: Molecular psychiatry

Article Title: Functional Analysis of Rare Variants Found in Schizophrenia Implicates a Critical Role for GIT1-PAK3 Signaling in Neuroplasticity

doi: 10.1038/mp.2016.98

Figure Lengend Snippet: (A) GIT1 structural and functional domains (see Introduction for description). Locations of unique GIT1 coding variants are shown above (SCZ cases; in red) or below (controls; in blue) the domain structure diagram. The SHD domain (Spa2 Homology Domain) is a key protein-protein interaction region known to mediate interactions with ARHGEF6/7-PAK, MAP2K1 (MEK1), FAK, PYK2, PLCO, and PPFIA2. (B) Summary of study design. Coding variants unique to SCZ cases or controls identified in large-scale human exome sequencing studies were tested in a range of biological assays in cell lines and primary cultured neurons to characterize and compare the effect of different variant effects and reveal potentially relevant functional changes. See also Figures S4, Table S1, and Table S2.

Article Snippet: anti-ARHGEF6 (Cell Signaling, #4573), anti-DLG2 (Neuro Mab, #73–284), anti-DLG4 (Neuro Mab, #73–028), anti-GABRG2 (Synaptic Systems, #224 003), anti-GAD1 (Chemicon, #MAB5406), anti-GAPDH (Cell Signaling, #2118), anti-GAPDH (Cell Signaling, #8884), anti-GIT1 (Cell Signaling, #2919), anti-GRIA1 (Millipore, #ABN241), anti-GRIA2 (Millipore, #AB1768), anti-GRIK2/3 (GLUR6/7) (Millipore, #04–921), anti-GRM5 (Millipore, #AB5675), anti-MEK1 (MAP2K1) (Cell Signaling, #9146), anti-NLGN1 (Neuro Mab, #73–158), anti-PAK1 (Cell Signaling, #2602), anti-PAK3 (Cell Signaling, #2609), anti-β-Tubulin (Sigma, #T8660), anti-SLC17A7 (VGLUT1) (Synaptic Systems, #135303).

Techniques: Functional Assay, Sequencing, Cell Culture, Variant Assay

Journal: Molecular psychiatry

Article Title: Functional Analysis of Rare Variants Found in Schizophrenia Implicates a Critical Role for GIT1-PAK3 Signaling in Neuroplasticity

doi: 10.1038/mp.2016.98

Figure Lengend Snippet: (A) Schematic diagram depicting the biochemical basis of the GIT1-PAK3 co-transfection assay. Rac/Cdc42 Guanine Nucleotide Exchange Factor (ARHGEF6/7), also known as the PIX (p21-activated kinase interacting exchange factor) family, bind to the PIX binding domain (PBD) on PAK family members in the presence of GIT1, leading to conformational changes and activation of PAKs. Deactivated PAK dimers are arranged in a head-to-tail manner in an auto-inhibited state due to interaction of one N-terminal Regulatory Domain (RD) composed of an auto-inhibitory kinase domain with the opposing C-terminal Kinase Domain (KD). As reviewed in (63), upon GTP-bound Rac/Cdc42 family GTPase binding to the GTPase Binding Domain (GBD), PAKs undergo a conformational change enabling autophosphorylation in trans that switches PAKs to an active state. Whereas auto-phosphorylation of S144 reduces the interaction of the kinase auto-inhibitory domain and kinase domain, auto-phosphorylation of S199/S204 reduces PAK--ARHGEF6/7 (PIX) interaction. Activation-loop phosphorylation at T423, which is required for full catalytic activity, occurs through autophosphorylation in trans or from the activity of other kinases. (B-D) Effect of wild-type GIT1 and GIT1-R283W on PAK3 and MAPK activation in HEK293 cells. Cells were co-transfected with the indicated expression vectors and 24hrs later cells were harvested in 2X SDS sample buffer. (B) Protein samples were subjected to immunoblot analysis using the indicated antibodies. For pMAPK, the top band corresponds to phospho-MAPK3 (ERK1; p44) T202/Y204 and the bottom band corresponds to phospho-MAPK1 (ERK2; p42) T185/T187. (C) Elevation of phospho-MAPK3, phospho-MAPK1 and Myc-PAK3 levels with wild-type GIT1 but not GIT1-R283W. For immunoblots other than those for the phospho-PAKs, band intensities were normalized to GAPDH band intensities, and then to values observed in wild-type GIT1-transfected cells in order to obtain fold changes (data represent mean + SEM; N = 3; **** and ** = p < 0.0001 and p < 0.01, respectively; two-way ANOVA with post hoc across row comparisons using a Tukey correction). (D) Elevation of phospho-PAK3 levels with wild-type GIT1 but not GIT1-R283W. For phospho-PAK immunoblots, band intensities were normalized to total Myc-PAK3 signals, and then to values for wild-type GIT1-transfected cells, in order to obtain fold changes (data represent mean + SEM; N = 3; **** = p < 0.0001; two-way ANOVA with post hoc across row comparisons using a Tukey correction). (E) GIT1-R283W shows reduced complex formation with PAK3, compared to wild-type GIT1. HEK293 cells were transfected with the indicated expression vectors. One day after transfection, cells were lysed and immunoprecipitated with FLAG-M2 antibody-conjugated agarose. The resulting FLAG-M2-immunoprecipitates were analyzed by immunoblot analysis with anti-FLAG-M2 or anti-Myc antibodies. (F) Quantitation of the decreased association between Myc-PAK3 and wild-type GIT1 or GIT1-R283W. Co-immunoprecipitated PAK3 immunoblot band intensities were normalized to immunoprecipitated GIT1-FLAG values. Data represent mean + SEM; N = 4; **** = p < 0.0001; two-tailed t-test; group variances were not significantly different. See also Figure S2.

Article Snippet: anti-ARHGEF6 (Cell Signaling, #4573), anti-DLG2 (Neuro Mab, #73–284), anti-DLG4 (Neuro Mab, #73–028), anti-GABRG2 (Synaptic Systems, #224 003), anti-GAD1 (Chemicon, #MAB5406), anti-GAPDH (Cell Signaling, #2118), anti-GAPDH (Cell Signaling, #8884), anti-GIT1 (Cell Signaling, #2919), anti-GRIA1 (Millipore, #ABN241), anti-GRIA2 (Millipore, #AB1768), anti-GRIK2/3 (GLUR6/7) (Millipore, #04–921), anti-GRM5 (Millipore, #AB5675), anti-MEK1 (MAP2K1) (Cell Signaling, #9146), anti-NLGN1 (Neuro Mab, #73–158), anti-PAK1 (Cell Signaling, #2602), anti-PAK3 (Cell Signaling, #2609), anti-β-Tubulin (Sigma, #T8660), anti-SLC17A7 (VGLUT1) (Synaptic Systems, #135303).

Techniques: Cotransfection, Binding Assay, Activation Assay, Activity Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5

doi: 10.1038/ncomms7137

Figure Lengend Snippet: ( a ) Immunoblotting analysis of GST-Pdlim5 pull-down assay. Eluates were subjected to immunoblotting with anti-Arhgef6 antibody. Coomassie staining demonstrates equal loading of GST-Pdlim5 proteins. ( b ) Immunostaining images of Arhgef6 and Pdlim5 from KDR/EGFP-Pdlim5 cells. Boxed area in KDR/EGFP-WT-Pdlim5 cell highlights representative co-localization of Arhgef6 with Pdlim5 at the cell periphery. Scale bars, 10 μm. ( c ) Immunostained images of Arhgef6 knockdown vSMCs stained with anti-Pdlim5 antibody, anti-Arhgef6 antibody and phalloidin. Scale bars, 10 μm.

Article Snippet: The following antibodies were purchased from the indicated suppliers: anti-AMPKα (1:2,000; Cell Signaling, 2603), phospho-Thr172 AMPKα (1:2,000; Cell Signaling, 2535), ACC (1:2,000; Cell Signaling, 3676), phospho-Ser79 ACC (1:2,000; Cell Signaling, 3661), anti-paxillin (1:1,000, Zymed Laboratories), anti-α-actinin (D6F6) (1:1,000 for immunoblot; Cell Signaling, 6487), monoclonal anti-α-actinin (1:1,000 for immunostain; Sigma-Aldrich, A5044), anti-Arp2 antibody (1:1,000; Cell Signaling, 3128), anti-Arp3 (FMS338) (1:500 for immunostaining; Abcam, ab49671), anti-Arpc2 (EPR8533) (1:2,000; Abcam, ab133315), anti-Arhgef6/Cool2/αPIX (C23D2) (1:1,000 for immunoblot; 1:400 for immunostain; Cell Signaling, 4573), anti-Gapdh antibody (1:5,000; Millipore, MAB374), anti-GFP-horseradish peroxidase (HRP) (1:3,000; MBL, 598-7), anti–RFP-HRP (1:3,000; MBL, PM005-7), anti-FLAG M2-HRP (1:5,000; Sigma-Aldrich, A8592), anti-V5-HRP antibody (1:5,000; Life Technologies, R961-25), HRP-coupled goat anti–rabbit (1:8,000; Cappel, 55696), anti-mouse IgG (1:8,000; Cappel, 55550), Alexa Fluor 488- (1:1,000 for staining; Life Technologies, A11029), Alexa Fluor 546- (1:1,000 for staining; Life Technologies, A11003) and Alexa Fluor 568-labelled secondary antibodies (1:1,000 for staining; Life Technologies, A11011), and Alexa Fluor 647 phalloidin (1:100 for staining; Cell Signaling, 8940).

Techniques: Western Blot, Pull Down Assay, Staining, Immunostaining

Journal: Nature Communications

Article Title: Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5

doi: 10.1038/ncomms7137

Figure Lengend Snippet: ( a ) Under normal conditions, Arhgef6 recruited to the IPP complex at the cell periphery activates Rac1, contributing to efficient lamellipodia formation. ( b ) Once AMPK activity is augmented, Ser177 of Pdlim5 is phosphorylated, displacing Arhgef6 from the IPP complex and causing attenuation of lamellipodia. Boxed areas at the cell periphery are expanded, representing views from the top (left) and from the side (right); see text for explanation. ILK, integrin-linked kinase; PINCH, particularly interesting new cysteine-histidine-rich protein; Pdlim5, PDZ and LIM domain 5; WAVE, Wiskott–Aldrich Syndrome protein family verprolin homologous.

Article Snippet: The following antibodies were purchased from the indicated suppliers: anti-AMPKα (1:2,000; Cell Signaling, 2603), phospho-Thr172 AMPKα (1:2,000; Cell Signaling, 2535), ACC (1:2,000; Cell Signaling, 3676), phospho-Ser79 ACC (1:2,000; Cell Signaling, 3661), anti-paxillin (1:1,000, Zymed Laboratories), anti-α-actinin (D6F6) (1:1,000 for immunoblot; Cell Signaling, 6487), monoclonal anti-α-actinin (1:1,000 for immunostain; Sigma-Aldrich, A5044), anti-Arp2 antibody (1:1,000; Cell Signaling, 3128), anti-Arp3 (FMS338) (1:500 for immunostaining; Abcam, ab49671), anti-Arpc2 (EPR8533) (1:2,000; Abcam, ab133315), anti-Arhgef6/Cool2/αPIX (C23D2) (1:1,000 for immunoblot; 1:400 for immunostain; Cell Signaling, 4573), anti-Gapdh antibody (1:5,000; Millipore, MAB374), anti-GFP-horseradish peroxidase (HRP) (1:3,000; MBL, 598-7), anti–RFP-HRP (1:3,000; MBL, PM005-7), anti-FLAG M2-HRP (1:5,000; Sigma-Aldrich, A8592), anti-V5-HRP antibody (1:5,000; Life Technologies, R961-25), HRP-coupled goat anti–rabbit (1:8,000; Cappel, 55696), anti-mouse IgG (1:8,000; Cappel, 55550), Alexa Fluor 488- (1:1,000 for staining; Life Technologies, A11029), Alexa Fluor 546- (1:1,000 for staining; Life Technologies, A11003) and Alexa Fluor 568-labelled secondary antibodies (1:1,000 for staining; Life Technologies, A11011), and Alexa Fluor 647 phalloidin (1:100 for staining; Cell Signaling, 8940).

Techniques: Activity Assay