Journal: The Journal of Biological Chemistry

Article Title: Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel

doi: 10.1074/jbc.RA119.011789

Figure Lengend Snippet: Pon3 reduces ENaC surface and whole-cell expression in FRT cells. A, FRT cells were transiently transfected with three ENaC subunits, where only the α subunit had an N-terminal HA tag and a C-terminal V5 tag. Equal amounts of the mouse Pon3 plasmid or the pCMV6 vector were cotransfected. Surface ENaC was labeled with biotin and recovered with NeutrAvidin beads at 4 °C. Blots were probed for the α subunit, γ subunit, Pon3, or GAPDH in the biotinylated surface fraction and in 5% of the total whole-cell lysates. B, the abundance of the α subunit (full-length 95 kDa and cleaved 30 kDa, indicated by arrowheads) or the γ subunit (∼75 kDa) in whole-cell lysate was normalized to the loading control (GAPDH) and expressed as the percentage in cells transfected with ENaC alone (−Pon3). C, the abundance of the α subunit (full-length 95 kDa and cleaved 30 kDa) or the γ subunit (∼75 kDa) at the cell surface was normalized to whole-cell abundance of the α or γ subunit, respectively, and then expressed as the percentage in cells transfected with ENaC alone (−Pon3). D, as a control, FRT cells were cotransfected with HAαV5βγ and with either γ-glutamyl transferase (+γGT) or an equal amount of the pCDNA3 vector (−γGT). Whole-cell lysates were collected and probed for both αENaC and γGT (indicated by the arrow). E, the abundance of the α subunit (95 kDa and 30 kDa) was normalized to the loading control (GAPDH) and expressed as the percentage in cells transfected with ENaC alone (−γGT). Experiments were repeated a total of three times with FRT cells of different passages. The summarized data are shown in a scatter-dot plot, with a horizontal bar indicating the mean. Statistical comparisons were analyzed with one-way ANOVA followed by Sidak's multiple comparisons test (**, p < 0.01; ***, p < 0.001).

Article Snippet: The recovered surface proteins and 5% of the total lysate were separated by SDS-PAGE and blotted for the α subunit with HA-HRP antibodies (0.05 μg/ml, 3F10, Sigma), the γ subunit (0.3 μg/ml, StressMarq), PON3 (0.03 μg/ml, Sigma), or GAPDH (0.3 μg/ml, Proteintech, HRP-60004), as described previously ( 29 , 106 ).

Techniques: Expressing, Transfection, Plasmid Preparation, Labeling

Journal: Scientific Reports

Article Title: Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle

doi: 10.1038/s41598-021-94709-0

Figure Lengend Snippet: Real-time PCR gene expression of Akt, p70S6K, 4E-BP1, and muscle-specific E3 ubiquitin ligases MAFbx and MuRF1. US unstretched, St stretched. Values are expressed as mean ± SEM. *P < 0.05, n = 8/group.

Article Snippet: After blocking with a blocking buffer (Boster Biological Technology, Pleasanton, CA, USA) for 1.5 h at room temperature, the membrane was incubated with primary antibodies (diluted in TBS-T) against p-p70S6K (Thr 389) (1:1,000; A0533; Assay Biotechnology Company, Sunnyvale, CA, USA) and p70S6K (1:1,000; CSB-PA003686; CUSABIO, Houston, TX, USA), p-4E-BP1 (Thr37/46) (1:1,000; #2855; Cell Signaling technology, Danvers, MA, USA) and 4E-BP1 (1:1000; CSB-PA007994; CUSABIO), p-AKT (S473) (1:1000; CSB-PA000466; CUSABIO), AKT (1:1000; CSB-PA008132; CUSABIO), GAPDH (1:1000; CSB-PA004666; CUSABIO) for 2 h at room temperature.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle

doi: 10.1038/s41598-021-94709-0

Figure Lengend Snippet: The phosphorylation levels of Akt, p70S6K, and 4E-BP1 measured with Western blotting. Representative western blot images and quantification of phosphorylation levels of ( A ) Akt, ( B ) p70S6K, and ( C ) 4E-BP1. US unstretched, St stretched. Values are expressed as mean ± SEM. *P < 0.05, n = 7/group.

Article Snippet: After blocking with a blocking buffer (Boster Biological Technology, Pleasanton, CA, USA) for 1.5 h at room temperature, the membrane was incubated with primary antibodies (diluted in TBS-T) against p-p70S6K (Thr 389) (1:1,000; A0533; Assay Biotechnology Company, Sunnyvale, CA, USA) and p70S6K (1:1,000; CSB-PA003686; CUSABIO, Houston, TX, USA), p-4E-BP1 (Thr37/46) (1:1,000; #2855; Cell Signaling technology, Danvers, MA, USA) and 4E-BP1 (1:1000; CSB-PA007994; CUSABIO), p-AKT (S473) (1:1000; CSB-PA000466; CUSABIO), AKT (1:1000; CSB-PA008132; CUSABIO), GAPDH (1:1000; CSB-PA004666; CUSABIO) for 2 h at room temperature.

Techniques: Phospho-proteomics, Western Blot

Journal: Scientific Reports

Article Title: Passive repetitive stretching is associated with greater muscle mass and cross-sectional area in the sarcopenic muscle

doi: 10.1038/s41598-021-94709-0

Figure Lengend Snippet: Sequences of real-time PCR primers used.

Article Snippet: After blocking with a blocking buffer (Boster Biological Technology, Pleasanton, CA, USA) for 1.5 h at room temperature, the membrane was incubated with primary antibodies (diluted in TBS-T) against p-p70S6K (Thr 389) (1:1,000; A0533; Assay Biotechnology Company, Sunnyvale, CA, USA) and p70S6K (1:1,000; CSB-PA003686; CUSABIO, Houston, TX, USA), p-4E-BP1 (Thr37/46) (1:1,000; #2855; Cell Signaling technology, Danvers, MA, USA) and 4E-BP1 (1:1000; CSB-PA007994; CUSABIO), p-AKT (S473) (1:1000; CSB-PA000466; CUSABIO), AKT (1:1000; CSB-PA008132; CUSABIO), GAPDH (1:1000; CSB-PA004666; CUSABIO) for 2 h at room temperature.

Techniques: Real-time Polymerase Chain Reaction