Journal: The Journal of Physiological Sciences : JPS

Article Title: Protein kinase C activation upregulates human L-type amino acid transporter 2 function

doi: 10.1186/s12576-021-00795-0

Figure Lengend Snippet: Effect of PKC activation on hLAT2 transport activity in S2-Mock and S2-LAT2 cells. a hLAT2 mRNA expression levels in S2-Mock and S2-LAT2 cells were examined by qPCR. The values were normalized to the mGapdh mRNA expression level, and the data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. b Functional expression of hLAT2 in S2-Mock and S2-LAT2 cells was examined by a transport assay. The cells were incubated with alanine (1 µM) in Na + -free HBSS buffer for 1 min at 37℃. BCH (1 mM) was used as a LAT inhibitor, and its solvent (0.1% (v/v) DMSO) was used as a control. Data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. **, p < 0.01; NS, not significant. c The effect of PKC activation on hLAT2 transport activity in S2-Mock and S2-LAT2 cells was examined by a transport assay. To examine the response of hLAT2 to PKC activation, the cells were treated in the absence (0.1% (v/v) DMSO, -) or presence of PMA (0.1 or 1 µM) for 30 min. To determine whether the PMA-induced effect depends on PKC activation, the cells were exposed to a pan-PKC inhibitor, Go6983 (10 µM), or its solvent (0.1% (v/v) DMSO, -) for 30 min at the same timing as the PMA treatment. After removing the reagents, the cells were incubated with alanine (1 µM) in Na + -free HBSS buffer for 1 min at 37℃. Data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. **, p < 0.01 vs -/- (PMA/Go6983), ##, p < 0.01 vs 0.1 µM/-, ††, p < 0.01 vs 1 µM/-

Article Snippet: Total RNA was isolated from the cells using Nucleospin RNA plus (MACHEREY–NAGEL, Düren, Germany) according to the manufacturer’s protocol. cDNA was synthesized from the extracted RNA (1 μg) using ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol.

Techniques: Activation Assay, Activity Assay, Expressing, Functional Assay, Transport Assay, Incubation, Solvent, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: Protein kinase C activation upregulates human L-type amino acid transporter 2 function

doi: 10.1186/s12576-021-00795-0

Figure Lengend Snippet: Effect of PKC activation on alanine uptake activity in Caco-2 cells. a hLAT1 and hLAT2 mRNA expression levels in Caco-2 cells were examined by qPCR. The values were normalized to the hGAPDH mRNA expression level, and the data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. b Functional expression of hLATs in Caco-2 cells was examined by a transport assay. The cells were incubated with alanine (50 µM) in Na + -free HBSS buffer for 5 min at 37℃. Overloaded alanine (10 mM) and BCH (1 mM) were used as inhibitors for alanine uptake-related transporters and LATs, respectively. Its solvent (0.1% (v/v) sterilized water) was used as a control (-). Data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. **, p < 0.01 vs control (-); NS, not significant. c , d The effect of PKC activation on alanine uptake activity in Caco-2 cells was examined by a transport assay. To activate PKC, the cells were treated in the absence (0.1% (v/v) DMSO, -) or presence of PMA (0.1 or 1 µM) for 4 h. To determine whether the PMA-induced effect depends on PKC activation, the cells were exposed to Go6983 (1 µM) or its solvent (0.1% (v/v) DMSO, -) at the same timing as the PMA treatment. After removing the reagents, alanine uptake was conducted in the same way as explained in b . d To elucidate the alanine uptake activity by hLAT1, an LAT1-specific inhibitor, JPH203 (10 µM), was added to the substrate solution. Data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. *, p < 0.05; **, p < 0.01 vs -/- (PMA/Go6983), ##, p < 0.01 vs 0.1 µM/-, †, p < 0.05; ††, p < 0.01 vs 1 µM/-

Article Snippet: Total RNA was isolated from the cells using Nucleospin RNA plus (MACHEREY–NAGEL, Düren, Germany) according to the manufacturer’s protocol. cDNA was synthesized from the extracted RNA (1 μg) using ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol.

Techniques: Activation Assay, Activity Assay, Expressing, Functional Assay, Transport Assay, Incubation, Solvent, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: Protein kinase C activation upregulates human L-type amino acid transporter 2 function

doi: 10.1186/s12576-021-00795-0

Figure Lengend Snippet: Effect of PKC activation on hLAT2 mRNA and protein expression levels. a The effect of PKC activation on hLAT2 mRNA expression levels in S2-Mock and S2-LAT2 cells was examined by qPCR. Total RNA was isolated from the cells that had been treated with DMSO (0.1% (v/v), -) or PMA (0.1 or 1 µM) for 30 min. The values were normalized to the mGapdh mRNA expression level, and the data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. NS, not significant. b The effect of PKC activation on hLAT2 protein expression levels in S2-Mock and S2-LAT2 cells was examined by Western blot analysis. Whole cell lysates were prepared from cells that had been treated in the same way as explained in a . β-Actin was used as a loading control, and representative results that were obtained from three independent experiments are shown. c The effect of PKC activation on hLAT2 mRNA expression levels in Caco-2 cells was examined by qPCR. Total RNA was isolated from the cells that had been treated with DMSO (0.1% (v/v)) or PMA (1 µM) for 4 h. The values were normalized to the hGAPDH mRNA expression level, and the data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. NS, not significant. d The effect of PKC activation on hLAT2 protein expression levels in Caco-2 cells was examined by Western blot analysis. Whole cell lysates were prepared from cells that had been treated in the same way as explained in c . β-Actin was used as a loading control, and representative results that were obtained from three independent experiments are shown

Article Snippet: Total RNA was isolated from the cells using Nucleospin RNA plus (MACHEREY–NAGEL, Düren, Germany) according to the manufacturer’s protocol. cDNA was synthesized from the extracted RNA (1 μg) using ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol.

Techniques: Activation Assay, Expressing, Isolation, Western Blot, Control