Journal: Physiological Reports

Article Title: Moderate alcohol consumption does not impair overload-induced muscle hypertrophy and protein synthesis

doi: 10.14814/phy2.12333

Figure Lengend Snippet: Muscle overload-induced increases in S6K1 substrates rpS6 and eEF2 are not prevented by alcohol consumption. The relative amount of phosphorylated and total rpS6 (A, B) and eEF2 (C, D) were measured in the plantaris muscle after 14 days of overload. Representative images of each marker correspond to the sample order depicted in the graph. * P < 0.05, indicates difference from Sham condition within that treatment. Values are expressed as means ± SE.

Article Snippet: Antibodies included (Cell Signaling, Beverly, MA, unless otherwise noted): S6K1, S6K1 (Thr 389 ), rpS6, rpS6 (Ser 240/244 ), 4E-BP1 (Bethyl Laboratories, Montgomery, TX), 4E-BP1 (Thr 37/46 ), eEF2, eEF2 (Thr 56 ), ERK1/2 (Thr 202 /Tyr 204 ), p42/44 MAPK, mTOR, mTOR (Ser 2448 ), REDD1 (ProteinTech, Chicago, IL), Akt, Akt (Thr 308 ), PRAS40, PRAS40 (Thr 246 ), ULK1, ULK1 (Ser 757 ), p62, LC3A/B, and GAPDH.

Techniques: Marker

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: A. Representative image of the overlaid images of Cy- labeled samples. B. Deep-Purple-stained gel image. The number 417 represented the protein spot of interest for EF2. C. The separation effect and protein content for EF2 in samples. The protein content was very high, and the separation effect was good. D. The expressions for protein spot EF2 in non-metastatic and metastatic LSCC tissues respectively, compared with para-carcinoma lung tissues of seven groups. SqNP, SqNT, SqMP and SqMT stand for non-metastastic para-carcinoma lung tissues, non-metastastic lung squamous cell carcinoma, metastastic para-carcinoma lung tissues and metastastic lung squamous cell carcinoma respectively. E. The Mascot score (65) of protein identification for spot 417 by MS. F. A representative tandem mass spectrum of the peptide (in red) matched for EF2 by MS.

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Labeling, Staining

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: A. Representative IHC images of the EF2 protein expression in LSCC specimens and matched peri-cacinoma lung tissues of tissue microarray. P and T stand for peri-cacinoma lung tissues and LSCC tissues respectively. B. Western blot analysis for EF2 in 16 pairs of LSCC tissues and peri-cacinoma lung tissues. The protein level of EF2 in LSCC tissues was 4.8-fold higher than that in the peri-cacinoma lung tissues (* p < 0.05).

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Expressing, Microarray, Western Blot

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: EF2 expression in LSCC and peri-cacinoma lung tissues of tissue microarray

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: Clinical characteristics and the expression for EF2 on the tissue microarray of LSCC

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Expressing, Microarray

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: A. Microscopy images under the phase contrast microscope. B. Microscopy images by crystal violet staining. NCI-H520/pcDB-EF2 cells had larger cell shapes with more elongated filopodia than NCI-H520/pcDB cells.

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Microscopy, Staining

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: A. CCK-8 assay. Transfection of EF2 significantly promoted the growth of NCI- H520/pcDB-EF2 cells. B. Colony formation assay. The ratio of colony formation of NCI-H520/pcDB-EF2 cells was much higher than that of NCI-H520/pcDB cells (* p < 0.05). C. FACS analysis. Cell cycle distribution was determined by the DNA content. A demonstrated decreased percentage in the G2/M phase of the NCI-H520/pcDB-EF2 cells (* p < 0.05). D. Expression of Akt1, p-Akt, p-Cylin B1, p-Cdc2, EF2 and β-actin detected by western blot.

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: CCK-8 Assay, Transfection, Colony Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: A. Cell scratch-wound assay. At 8 h after the scratching, few cells migrated across the wound in the NCI-H520/pcDB cell groups compared with the NCI-H520/ pcDB-EF2 cell groups. At 24 h, NCI-H520/pcDB-EF2 cells reached the midline of the wound and exhibited a faster migration rate than the control group (* p < 0.05). B. Transwell migration. Significantly more NCI-H520/ pcDB-EF2 cells migrated through the basement membrane compared with NCI-H520/pcDB cells (* p < 0.05). C. Transwell invasion. Significantly more NCI-H520/pcDB-EF2 cells invaded the basement membrane compared with NCI-H520/pcDB cells (* p < 0.05).

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Scratch Wound Assay Assay, Migration, Control, Membrane

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: A. The expression of β-catenin, E-cadherin, MMP2, Vimentin, EF2 and β-actin by western blot. B. The expressions of SNAIL , TWIST and FOXC1 detected by qPCR in three groups of the cells (* p <0.05). C. Representative IHC images of E-cadherin, Vimentin and MMP2 in EF2 positive clinical 22 pairs of LSCC and non-neoplastic peritumoral parts.

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

doi: 10.18632/oncotarget.11298

Figure Lengend Snippet: In nude mice, the NCI-H520/pcDB-EF2 cells grew larger tumors compared with the control group.

Article Snippet: Approximately 50 μg of protein from each of the 16 pairs of LSCC tissues with the non-neoplastic peritumoral parts were resolved in 8% SDS-PAGE (Bio-Rad, USA) After gel electrophoresis and membrane transfer, the membrane was probed with rabbit anti-EF2 monoclonal antibody (1:1000, EPITOMICS, China) and mouse anti-actin monoclonal antibody (1:3000, Proteintech Group, China).

Techniques: Control

Journal: Molecular Biology of the Cell

Article Title: Role of clathrin in dense core vesicle biogenesis

doi: 10.1091/mbc.E16-10-0742

Figure Lengend Snippet: Clathrin depletion from PC12 cells. (A) Cells were transduced with SMARTvector lentiviral shRNA targeting clathrin heavy chain (CHC). Infected cells were initially selected with puromycin. Then, because the shRNA is inducibly coexpressed with GFP, treatment with doxycycline overnight was used to induce expression. The doxycycline was then removed and high expressors were isolated by flow cytometry to provide a stable population of cells harboring inducible CHC shRNA. (B) To investigate the effects of clathrin depletion, the cells were either mock-treated or treated for 5 d with doxycycline. Immunofluorescence and Western blotting both show a robust loss of clathrin. EF2 was used as a loading control. Scale bar: 10 μm.

Article Snippet: Antibodies used in this study include an in-house rabbit polyclonal antibody against clathrin heavy chain ( Simpson et al ., 1996 ), goat antibodies against EF2 and CgB (Santa Cruz Biotechnology), and rat monoclonal anti-RFP (5F8, ChromoTek).

Techniques: Transduction, shRNA, Infection, Expressing, Isolation, Flow Cytometry, Immunofluorescence, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Role of clathrin in dense core vesicle biogenesis

doi: 10.1091/mbc.E16-10-0742

Figure Lengend Snippet: Effect of clathrin depletion on different types of vesicles. (A) Mass spectrometry was carried out on SILAC-labeled mock-treated and clathrin-depleted cell homogenates and vesicle-enriched fractions to identify proteins with altered behavior. More than 2500 proteins were quantified, of which a few examples are shown here. CCV-associated proteins (shown in red) were depleted from the vesicle-enriched fraction, but with the exception of clathrin they were not depleted from the cell homogenate. DCV-enriched proteins (shown in blue) were depleted 1.5- to 2-fold from the cell homogenate and 4- to 6-fold from the vesicle-enriched fraction, while SMV proteins (green) were unaffected in the homogenate but moderately depleted from the vesicle-enriched fraction. Other proteins (shown in black) were generally unaffected. Proteins are indicated by their gene names (Cltc = clathrin heavy chain; Clint1 = epsinR; Ap1m1 = AP-1 μ1; Scg2 = secretogranin II; Chgb = chromogranin B; Ptprn = receptor-type tyrosine-protein phosphatase-like N; Syp = synaptophysin; Sv2a = synaptic vesicle glycoprotein 2A; Vamp2 = VAMP2; Psma4 = proteasome subunit alpha 4; Tubb2a = tubulin beta 2A; Eef2 = eukaryotic translation elongation factor 2). Error bars show SD ( n = 3). (B) Principal component analysis combining the fractionation profiling data shown in and the data shown above on fold-depletion in the vesicle-enriched fraction after clathrin knockdown (three replicates). CCVs and DCVs are well separated, and new DCV components can be predicted, such as Emilin (circled). The identities of the protein are shown in Supplemental Figure S2.

Article Snippet: Antibodies used in this study include an in-house rabbit polyclonal antibody against clathrin heavy chain ( Simpson et al ., 1996 ), goat antibodies against EF2 and CgB (Santa Cruz Biotechnology), and rat monoclonal anti-RFP (5F8, ChromoTek).

Techniques: Mass Spectrometry, Labeling, Fractionation