Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: ( A ) Knockdown of eEF-2K by siRNA. siRNA specifically targeting eEF-2K inhibits its expression in MDA-MB-231 cells. Cells were transfected with eEF-2K siRNA (5–75 nM) for 48 h, and cell lysates were subjected to Western blot analysis. ( B ) Effect of knockdown of eEF-2K on cell proliferation. MDA-MB-231 cells were transfected with eEF-2K siRNA, and after 48 h proliferation was detected by an MTS assay. ( C ) Overexpression of eEF-2K in MDA-MB-231 cells. Cells were transiently transfected with an expression vector encoding GST-tagged wild type eEF-2K, and cell lysates (48 h) were subjected to Western blot analysis. For the effect of overexpression of eEF-2K on cell proliferation, MDA-MB-231 cells were transiently transfected with eEF-2K or a vector control, and cell proliferation was measured using an MTS assay after 96 h. ( D–E ) Effect of eEF-2K knockdown on colony formation. Knockdown of eEF-2K by siRNA (50 nM) significantly inhibited the number of colonies formed by MDA-MB-231 (D) and MCF-7 (E) cells (*p<0.05). Cells were transfected every 4 days with control or eEF-2K siRNA.

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Knockdown, Expressing, Transfection, Western Blot, MTS Assay, Over Expression, Plasmid Preparation, Control

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: Cells were transiently transfected with eEF-2K siRNA or GST-eEF-2K plasmid, and cell lysates (72 h) were subjected to Western blot analysis. ( A–B ) Knockdown of eEF-2K by siRNA increases expression levels of p27 Kip1 (A), while decreasing cyclin D1 (A) and c-Myc (B) levels. ( C–D ) eEF-2K knockdown additionally inhibits the activity of Src and FAK as indicated by reduced p-Src (Tyr-416) (C) and p-FAK (Tyr-397) (D). ( E ) Conversely, overexpression of eEF-2K resulted in increased p-Src (Tyr-416) levels. ( F ) Knockdown of eEF-2K also inhibits the activity of Akt as indicated by reduced p-Akt (Ser-473).

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Knockdown, Expressing, Activity Assay, Over Expression

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: ( A ) Treatment schedule of nude mice bearing MDA-MB-231 tumors with liposomal siRNA (control and eEF-2K) and/or doxorubicin. About 2 weeks after tumor cell injection (MDA-MB-231 cells), targeting of eEF-2K was achieved by systemic ( i.v. from tail vein) administration of DOPC-liposomal eEF-2K siRNA (150 µg/kg or about 4 µg/mouse) twice a week and/or doxorubicin once a week ( i.p. , 4 mg/kg), for 4 weeks. ( B ) Tumor weight of L-Control and L-eEF-2K siRNA treated mice. Therapeutic targeting of the eEF-2K gene was achieved by systemically administering ( i.v. twice a week, 150 µg/kg) DOPC-liposomal siRNA (L-eEF-2K siRNA#1 and siRNA#2) in nude mice bearing MDA-MB-231 tumors. Non-silencing DOPC-liposomal siRNA (L-Control siRNA) was used as a control. After 4 weeks of treatment, tumor weight was measured. ( C ) The mean weight of mice after four weeks of treatment. No toxic effects were observed in mice exposed to liposomal eEF-2K siRNA ± doxorubicin for four weeks, compared with the control group. Mice appeared healthy and did not lose weight during treatment. ( D–E ) TUNEL assay indicating apoptosis (D) and quantification (% apoptosis) of TUNEL assay (E) in tumors in mice treated with L-eEF-2K siRNA and/or doxorubicin. Brown staining in the TUNEL assay is a positive indicator of apoptosis. ( F ) In vivo targeting of eEF-2K enhances the efficacy of chemotherapy. Mice bearing MDA-MB-231 tumors were given L-eEF-2K siRNA or L-Control siRNA ( i.v. twice a week, 150 µg/kg) and doxorubicin once a week ( i.p. , 4 mg/kg) for 4 weeks, after which tumor weights were measured.

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Injection, TUNEL Assay, Staining, In Vivo

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: Following treatment with L-eEF-2K siRNA, tumor tissues were removed from mice and subjected to Western blot analysis. ( A–B ) Treatment of mice with L-eEF-2K siRNA results in the knockdown of eEF-2K in different tumors (A–B), with the consequent reduction in p-eEF2 (Thr-56) levels (B). ( C ) L-eEF-2K siRNA treatment induces apoptosis in tumors as indicated by caspase-9 cleavage and a decrease in anti-apoptotic Bcl-2 levels. ( D ) Depletion of eEF-2K enhances doxorubicin-induced Bcl-2 and HIF1α down-regulation.

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot, Knockdown

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: ( A ) Western blot analysis demonstrated that knockdown of eEF-2K in doxorubicin-treated MDA-MB-231 cells further reduced cyclin D1 levels while increasing p27 Kip1 levels. ( B ) Knockdown of eEF-2K using two different siRNA enhances efficacy of doxorubicin-induced (0.1 and 0.5 ìM) apoptosis of MDA-MB-231 breast cancer cells. Apoptosis was detected by Annexin V staining and the percentage of positive cells was quantified by FACS analysis after 48 h of doxorubicin treatment. ( C ) Down-regulation of eEF-2K enhances inhibition of Src and FAK activity and increases caspase-9 cleavage in doxorubicin-treated MCF-7/DoxR cells.

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot, Knockdown, Staining, Inhibition, Activity Assay

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: Depletion of eEF-2K by siRNA inhibits invasion (A), while overexpression of eEF-2K increases invasion (B) of MDA-MB-231 cells in Matrigel (72 h).

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Over Expression

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: Down-regulation of eEF-2K by siRNA enhances the efficacy of paclitaxel-induced (2 nM) inhibition of colony formation in highly aggressive and metastatic MDA-MB-231 breast cancer cells. ( A ) Cell colonies stained with crystal violet and ( B ) Quantification of colony formation represented graphically.

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Inhibition, Staining

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: The phosphorylation of eEF2 by eEF-2K leads to an increase in the phospho-eEF2/eEF2 ratio in transformed breast cancer cells, which is predicted to influence protein translation, possibly facilitating the selective translation of mRNAs possessing structured 5′ -UTRs encoding for proteins such as c-Myc, Bcl-2 and cyclin D1. eEF-2K facilitates basal c-Src activation through a mechanism that is currently unknown. c-Src is an important mediator of many downstream effects of receptor tyrosine kinases, including the EGFR family, and mediates basal activation of Akt by eEF-2K. Pathways facilitated by eEF-2K are implicated in cell growth, survival, motility and angiogenesis, supporting the notion that it may be a potential cancer therapeutic target. Known mechanisms are represented as solid arrows. Potential mechanisms are represented as dashed arrows.

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Phospho-proteomics, Transformation Assay, Activation Assay

Journal: Oncotarget

Article Title: FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells

doi: 10.18632/oncotarget.7672

Figure Lengend Snippet: Cells were transfected with 50 nM FOXM1#1 or FOXM1#2 siRNA or control siRNA. Protein extracts were isolated 72 h after transfection. β1-Actin was used as a loading control. ( A , B ) Knockdown of FOXM1 by siRNA decreased expression levels of Cyclin D in both cell lines. FOXM1 knockdown additionally by siRNA reduced expression levels of p-ERK (Thr-202) in BT-20 cells but not in MDA-MB-231 cells. The expression level of ERK was not changed in either MDA-MB-231 or BT-20 cells. Knockdown of FOXM1 also inhibited p-eEF2 levels (Thr-56), but the expression level of eEF2 was not changed in either MDA-MB-231 or BT-20 cells. ( C , D ) Knockdown of FOXM1 suppressed the protein levels of eEF2K and p-Src (Tyr-416) but not Src in both cell lines.

Article Snippet: The membranes were blocked with a blocking buffer (0.1 Triton X-100 with 5% dry milk in Tris-buffered saline–Tween 20 [TBS-T]) for 60 min. After being washed with TBS-T, the membranes were probed with the following primary antibodies: FOXM1 (C-20, Santa Cruz Biotechnology, Dallas, TX), extracellular signal-regulated kinase (ERK), and p-ERK (Thr-202) (Santa Cruz Biotechnology, Dallas, TX) and eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, Src, and p-Src (Tyr-416) (Cell Signaling Technology, Danvers, MA).

Techniques: Transfection, Control, Isolation, Knockdown, Expressing

Journal: bioRxiv

Article Title: YBX3 overexpression in mesothelioma drives aberrant cell proliferation

doi: 10.64898/2026.02.11.705262

Figure Lengend Snippet: (A) Cell proliferation profiles from the IncuCyte Live-Cell instrument. MpM cells were treated for 24 hours with siRNA specific for YBX3 or non-targeting control before monitoring growth during the following 72 hours. Error bars represent means ± SD (n = 3 independent experiments). Significance assed by unpaired Student’s t test (* = p<0.05 and ** = p<0.01). ( B ) In parallel with (A), representative western blot of YBX3 protein expression levels confirming the knockdown efficiency at 72 hours. ( C ) MpM cell lines 8T and 13T cells transfected with siRNA specific for YBX3 or non-targeting control were pulse-labelled with [35S]-methionine for 30 min. Total counts per minute were normalized to total protein, and values are shown as a fold change for each cell line. Error bars represent means ± SD (n = 3 independent experiments). Significance assed by unpaired Student’s t test (* = p<0.05 and ** = p<0.01). ( D ) Comparison of polysome profiles from MpM cell lines transfected with siRNA specific for YBX3 or non-targeting control for 72 hours. Cytoplasmic lysates were centrifuged at 38,000 rpm through 10 to 50% sucrose gradients at 4°C for 2 hours, and absorbance was measured at 254 nm using a flow rate of 1 ml/min. Profiles shown were representative of two independent experiments. ( E ) Representative western blots from 8T MpM cells treated with siRNA specific for YBX3 or non-targeting control for the indicated times. Cells were lysed and analysed by immunoblotting with the indicated antibodies. ( F ) Quantification of eEF2 (T-56) and eIF2α (S-51) phosphorylation from (F). Error bars represent means ± SD (n = 3 independent experiments). Significance assed by unpaired Student’s t test (* = p<0.05).

Article Snippet: Primary antibodies used were DBPA (YBX3) (#NBP1-71827, Novus), LAT1 (#5347, CST), eEF2 (#sc-166415, Santa Cruz), p-eEF2 (Thr56) (#2331, CST), eIF2α (#9722, CST), p-eIF2α (Ser51) (#109202, Abcam), p–4E-BP1 (Ser65) (#9451,CST), 4E-BP1 (9644, CST), p–p70 S6K (Thr389) (#9205, CST), p70 S6K (#2708, CST), β-actin (#A5441, Sigma), DDX3 (#8192, CST), LAT1 (#5347, CST) SLC3A2 (#HPA017980, Atlas antibodies) and β-tubulin (#2146, CST).

Techniques: Control, Western Blot, Expressing, Knockdown, Transfection, Comparison, Phospho-proteomics

Journal: bioRxiv

Article Title: YBX3 overexpression in mesothelioma drives aberrant cell proliferation

doi: 10.64898/2026.02.11.705262

Figure Lengend Snippet: (A) Abundance of amino acids in 8T and 13T MpM cell lines treated with JPH203 (10 µM) for the indicated times using LC-MS. Heatmap shows mean relative abundance (n = 6 independent biological replicates per group). (B) Representative western blots from 8T cells were treated with JPH203 (10 µM) or DMSO for the indicated times. (C) Quantification of eEF2 (T-56) and eIF2α (S-51) phosphorylation from (B). Error bars represent means ± SD (n = 3 independent experiments). Significance assed by unpaired Student’s t test (* = p<0.05 and ** = p<0.01). (D) MpM cell lines 8T and 13T cells treated with JPH203 (10 µM) or DMSO for 6 hours were pulse-labelled with [35S]-methionine for 30 min. Total counts per minute were normalized to total protein and values are shown as a fold change for each cell line. Error bars represent means ± SD (n = 3 independent experiments) and significance assed by unpaired Student’s t test (* = p<0.05 and ** = p<0.01). (E) Comparison of polysome profiles from MpM 8T and 13T cell lines treated with JPH203 (10 µM) or DMSO for 6 hours. Cytoplasmic lysates were centrifuged at 38,000 rpm through 10 to 50% sucrose gradients at 4°C for 2 hours, and absorbance was measured at 254 nm using a flow rate of 1 ml/min. Profiles shown were representative of two independent experiments.

Article Snippet: Primary antibodies used were DBPA (YBX3) (#NBP1-71827, Novus), LAT1 (#5347, CST), eEF2 (#sc-166415, Santa Cruz), p-eEF2 (Thr56) (#2331, CST), eIF2α (#9722, CST), p-eIF2α (Ser51) (#109202, Abcam), p–4E-BP1 (Ser65) (#9451,CST), 4E-BP1 (9644, CST), p–p70 S6K (Thr389) (#9205, CST), p70 S6K (#2708, CST), β-actin (#A5441, Sigma), DDX3 (#8192, CST), LAT1 (#5347, CST) SLC3A2 (#HPA017980, Atlas antibodies) and β-tubulin (#2146, CST).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Phospho-proteomics, Comparison

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: Cells were transiently transfected with eEF-2K siRNA or GST-eEF-2K plasmid, and cell lysates (72 h) were subjected to Western blot analysis. ( A–B ) Knockdown of eEF-2K by siRNA increases expression levels of p27 Kip1 (A), while decreasing cyclin D1 (A) and c-Myc (B) levels. ( C–D ) eEF-2K knockdown additionally inhibits the activity of Src and FAK as indicated by reduced p-Src (Tyr-416) (C) and p-FAK (Tyr-397) (D). ( E ) Conversely, overexpression of eEF-2K resulted in increased p-Src (Tyr-416) levels. ( F ) Knockdown of eEF-2K also inhibits the activity of Akt as indicated by reduced p-Akt (Ser-473).

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Knockdown, Expressing, Activity Assay, Over Expression

Journal: PLoS ONE

Article Title: Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

doi: 10.1371/journal.pone.0041171

Figure Lengend Snippet: ( A ) Western blot analysis demonstrated that knockdown of eEF-2K in doxorubicin-treated MDA-MB-231 cells further reduced cyclin D1 levels while increasing p27 Kip1 levels. ( B ) Knockdown of eEF-2K using two different siRNA enhances efficacy of doxorubicin-induced (0.1 and 0.5 ìM) apoptosis of MDA-MB-231 breast cancer cells. Apoptosis was detected by Annexin V staining and the percentage of positive cells was quantified by FACS analysis after 48 h of doxorubicin treatment. ( C ) Down-regulation of eEF-2K enhances inhibition of Src and FAK activity and increases caspase-9 cleavage in doxorubicin-treated MCF-7/DoxR cells.

Article Snippet: The membranes were blocked with 5% dry milk or BSA and probed with the following primary antibodies: eEF-2K, p-EF2 (Thr-56), EF2, cyclin D1, p27, p-Akt (Ser-473), Akt, p-Src (Tyr-416), Src, p-paxillin (Tyr-31), p-mTOR (Ser-2448), mTOR (Cell Signaling Technology, Danvers, MA); HIF1α, p-FAK (Tyr-397), FAK (BD Transfection); c-Myc, Bcl-2 and caspase-9 (cleaved) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot, Knockdown, Staining, Inhibition, Activity Assay

Journal: Oncotarget

Article Title: MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase

doi: 10.18632/oncotarget.14264

Figure Lengend Snippet: A . The silencing of eEF2K by siRNA (50 nM) significantly reduced the number of colonies formed by MDA-MB-231 and MDA-MB-436 cells (*** p < 0.001). Cells were transfected every 4 days with the control or eEF2K siRNA. B . MDA-MB-231 and MDA-MB-436 cell invasion was assessed by using a matrigel transwell assay. Cells were transfected with eEF2K siRNA or control siRNA or not transfected (NT)); after 72-h transfection, the cells were transferred to transwell and incubated for 24 h and cells invading matrigel and passing through the membrane we counted by light microscope. C . Western blot analysis of p-EF2 Thr56 , p-Src Tyr416 , total Src, p-Fak Tyr397 , total Fak, p-Akt Ser473 , total Akt and cyclin D1 in the indicated cells. β-Actin was used as a loading control.

Article Snippet: The expression levels of selected proteins were detected by using specific antibodies for eEF2K, p-EF2 (Thr56), Src, p-Src ( Tyr416),(Cell Signaling Technology), FAK, p-FAK (Try397) (Thermo), p-AKT (Ser473), AKT, cyclin D1 (Santa Cruz) and β-actin (Sigma) and the corresponding HRP-conjugated secondary antibodies [ ].

Techniques: Transfection, Control, Transwell Assay, Incubation, Membrane, Light Microscopy, Western Blot