anti lat1 rabbit polyclonal antibody  (Novus Biologicals)

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: LAT1 knockdown delays mitotic progression via activation of spindle assembly checkpoint . HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). A , at 48 h after transfection, Western blot analysis was performed with the indicated antibodies. B , at 48 h after transfection, the HeLa S3 cells were fixed and stained for phospho-Histone H3 (pS10) and DNA. The mitotic indices are plotted as the mean ± SD of three independent experiments (n > 1000). Statistical analysis was performed using one-way ANOVA ( F = 10.37, p = 0.011), and asterisks indicate significant differences (Dunnett’s test, ∗ p < 0.05). C–F , At 28 h after siRNA transfection, the HeLa S3 cells were treated with 6 μM RO-3306 for 20 h, washed with PBS(+), and cultured for a further 75 min. The cells were then fixed and stained for α-tubulin ( green ) and DNA ( red ). C , a schematic depiction of the synchronization method is shown. D , representative images are shown. Arrows indicate the cells during cytokinesis. Scale bar, 20 μm. The mitotic cells were classified into four groups (see “ ”). The percentages of cells of the each group ( E ) or the percentage of mitotic cells ( F ) are plotted as the mean ± SD of three independent experiments [n > 200 in panel ( E ), n > 1000 in panel ( F )]. Statistical analysis was performed using one-way ANOVA [ F = 25.56, p = 0.001 in panel ( E ); F = 1.936, p = 0.224 in panel ( F )], and asterisks indicate significant differences (Dunnett’s test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, N.S., not significant). G and H , At 28 h after siRNA transfection, the HeLa S3 cells were treated with 6 μM RO-3306, washed, and cultured with or without 2 μM AZ-3146 treatment for a further 75 min. The percentages of cells of the each group ( G ) or the percentage of mitotic cells ( H ) are plotted as the mean ± SD of three independent experiments [n > 200 in panel ( G ), n > 1000 in panel ( H )]. Statistical analysis was performed using two-way ANOVA in panel ( G ) (siRNA, F = 43.447, p = 0.000; AZ3146, F = 55.156, p = 0.000; interaction, F = 1.473, p = 0.259) and in panel ( H ) (siRNA, F = 0.005, p = 0.948; AZ3146, F = 0.432, p = 0.529; interaction, F = 0.012, p = 0.917). Asterisks indicate significant differences (Tukey’s test, ∗∗ p < 0.01, N.S., not significant).

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Knockdown, Activation Assay, Transfection, Control, Western Blot, Staining, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: LAT 1 supports proper mitotic progression in an amino acid transport activity-independent manner . A and B , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2), or treated with 30 μM JPH203 for 48 h. The amino acid transport ability was evaluated using boronophenylalanine (BPA) and its probe (see “ ”). A , representative images are shown. Images of the BPA–probe fluorescence were pseudocolored as red. Scale bar, 100 μm. B , the fluorescence intensity of the BPA–probe fluorescent compound was measured and plotted as the mean ± SD from a representative experiment (n = 60). Statistical analysis was performed using Welch’s ANOVA ( F = 413, p = 0.000), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). C , HeLa S3 cells were treated with 30 μM JPH203 for 48 h, and the cells were monitored for a further 12 h by time-lapse imaging with 0.1 μM Hoechst 33342. The duration of each mitotic phase is presented: prophase/prometaphase (P/PM: chromosome condensation, green ), metaphase (M: chromosome alignment, red ), and anaphase/telophase (A/T: from anaphase onset to cleavage furrow ingression, blue ). In total, 40 mitotic cells were examined. D–F , doxycycline (Dox)-inducible LAT1-WT or LAT1-W257A (HeLa S3/LAT1-WT or LAT1-W257A) cell lines were transfected with siControl or siLAT1#2 with or without 2 μg/ml Dox. At 48 h after transfection, Western blot analysis was performed ( D ), and the amino acid transport ability was evaluated ( E , F ). E , representative images are shown. Images of the BPA–probe fluorescence were pseudocolored as red. Scale bar, 100 μm. F , The fluorescence intensity of the BPA–probe fluorescent compound was measured and plotted as the mean ± SD from a representative experiment (n = 60). Statistical analysis was performed using Welch’s ANOVA ( F = 160.7, p = 0.000 in the left panel ; F = 576, p = 0.000 in the right panel ), and asterisks indicate significant differences (Games–Howell test, ∗∗ p < 0.01; ∗∗∗ p < 0.001). G and H , HeLa S3/LAT1-WT or LAT1-W257A cells were transfected with siControl or siLAT1#2 with or without 2 μg/ml Dox treatment. At 28 h after siRNA transfection, the cells were treated with 6 μM RO-3306 for 20 h with or without Dox treatment, washed, and cultured for a further 90 min. The cells were then stained for α-tubulin and DNA. The percentages of cells of the each group (G) or the percentage of mitotic cells (H) are plotted as the mean ± SD of four independent experiments [n > 200 in panel ( G ), n > 1000 in panel (H)]. Statistical analysis was performed using one-way ANOVA in panel ( G ) ( F = 61.12, p = 0.000 in the left panel ; F = 33.17, p = 0.000 in the right panel ) and ( H ) ( F = 0.082, p = 0.922 in the left panel ; F = 0.537, p = 0.61 in the right panel ). Asterisks indicate significant differences (Tukey’s test, ∗∗ p < 0.01; ∗∗∗ p < 0.001; N.S., not significant).

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Activity Assay, Transfection, Control, Fluorescence, Imaging, Western Blot, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: LAT1 knockdown prolongs metaphase duration . A and B , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). At 19 h after siRNA transfection, the cells were treated with 4 mM thymidine for 20 h, washed with PBS(−), and cultured for a further 9 h. The cells were then monitored for 12 h by time-lapse imaging with 0.1 μM Hoechst 33342. A , images show the typical phenotypes of siRNA-treated mitotic cells: cells that exhibit normal mitosis (normal mitosis), the prolonged duration of metaphase (prolonged), and spindle misorientation (misorientation). Scale bar, 10 μm. B , the graphs are shown as indicated in Fig. 2 C . ∗, misorientation; #, cell death. In total, 40 mitotic cells were examined. C and D , MIA PaCa-2 cells were transfected with siControl or siLAT1#2. C , at 48 h after transfection, Western blot analysis was performed with the indicated antibodies. D , at 48 h after siRNA transfection, the cells were monitored for 12 h by time-lapse imaging with 0.1 μM Hoechst 33342. The graphs are shown as indicated in panel ( B ), and orange bars indicate the mitotic exit without chromosome segregation (slippage).

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Knockdown, Transfection, Control, Cell Culture, Imaging, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: LAT1 supports the spindle orientation with proper confinement of the lateral cortex localization of NuMA in a transport activity-independent manner . A–E , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). At 28 h after siRNA transfection, the cells were treated with 6 μM RO-3306 for 20 h, washed, and cultured for a further 60 min. A–C , the cells were fixed and stained for γ-tubulin ( green ) and DNA ( red ). A , Z-section images at 0.32-μm intervals (Z-axis) of a representative cell are shown from the lowest (0 μm) to the highest (4.5 μm) position. Scale bar, 10 μm. B , a schematic depiction of the metaphase cells is shown. The vertical distance (v) indicates the difference between the heights of two centrosomes within a cell. C , the difference in the height of γ-tubulin within a single cell was measured and plotted as the mean ± SD (n = 30). Statistical analysis was performed using Welch’s ANOVA ( F = 15.45, p = 0.000), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). D and E , the cells were fixed and stained for NuMA (gray) and DNA. D , representative images are shown. Scale bar, 10 μm. Dashed lines indicate NuMA localization at the cortex. The cells were classified into three groups (see “ ”). E , the percentages of cells of each group are plotted as the mean ± SD of three independent experiments (n > 100). Statistical analysis was performed using one-way ANOVA [ F = 32.08, p = 0.000 (lateral); F = 55.34, p = 0.000 (lateral + center)], and asterisks indicate significant differences (Dunnett’s test, ∗∗ p < 0.01; ∗∗∗ p < 0.001). F and G , HeLa S3/LAT1-WT or LAT1-W257A cells were transfected with siControl or siLAT1#2 with or without 2 μg/ml Dox. At 28 h after siRNA transfection, the cells were treated with 6 μM RO-3306 for 20 h with or without Dox, washed, and cultured for a further 60 min. The cells were then fixed and stained for γ-tubulin ( F ) or NuMA ( G ). F , the difference in the height of γ-tubulin within a single cell (vertical distance) was measured and plotted as the mean ± SD (n = 50). Statistical analysis was performed using Welch’s ANOVA ( F = 16.05, p = 0.000 in the left panel; F = 26.03, p = 0.000 in the right panel), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). G , the percentages of cells of each group are plotted as the mean ± SD of three independent experiments (n > 100). Statistical analysis was performed using one-way ANOVA in the left panel [ F = 105.6, p = 0.000 (lateral); F = 47.26, p = 0.000 (lateral + center)] and in the right panel [ F = 23.52, p = 0.001 (lateral); F = 14.09, p = 0.005 (lateral + center)]. Asterisks indicate significant differences (Tukey’s test, ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Activity Assay, Transfection, Control, Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: Plasma membrane-localized LAT1 is dispensable for mitotic progression . A , HeLa S3 cells were treated with 5 μM of S-Trityl-L-cysteine (STLC) for 16 h, and mitotic cells were collected by mitotic shake-off. Whole-cell lysates from asynchronous cells and mitotic cells were prepared in the presence or absence of 2-mercaptoethanol (2-ME) (see “ ”). Western blot analysis was performed with the indicated antibodies. B , HeLa S3/LAT1-WT cells were treated with 2 μg/ml Dox for 32 h and then treated with 5 μM STLC for a further 16 h to induce arrest at mitosis. Mitotic cells were collected by mitotic shake-off. LAT-WT was immunoprecipitated with an anti-HA antibody in the absence of 2-ME and subjected to Western blot analysis using the indicated antibodies. C , HeLa S3 cells were transfected with control siRNA (siControl) or CD98-targeting siRNAs (siCD98#1 and #2). At 48 h after transfection, Western blot analysis was performed with the indicated antibodies. D and E , HeLa S3/LAT1-WT cells transfected with siControl or siCD98#1 were cultured for 48 h in the presence of 1 μg/ml Dox treatment during the last 24 h. The cells were then fixed and stained for HA ( green ) and DNA ( red ). D , representative images are shown. Scale bar, 10 μm. E , the percentage of cells exhibiting LAT1 localization at the plasma membrane is plotted (n > 50). F , HeLa S3 cells were transfected with siControl, siCD98#1, or siCD98#2. At 48 h after siRNA transfection, the cells were monitored for 8 h by time-lapse imaging with 0.1 μM Hoechst 33342. The graphs are shown as indicated in Fig. 2 C .

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Membrane, Western Blot, Immunoprecipitation, Transfection, Control, Cell Culture, Staining, Imaging

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: LAT1 promotes Golgi unlinking along with Aurora A recruitment to the centrosomes . A , HeLa S3 cells were fixed with formaldehyde and stained for LAT1 ( green ) and TGN46 ( red ) or calnexin ( red ). Representative images are shown. Scale bar, 10 μm. B–E , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). At 19 h after siRNA transfection, the cells were treated with 4 mM thymidine for 19 h and washed with PBS(−). B and C , the cells were cultured for a further 9 h or 10 h in siControl or siLAT1 cells, respectively, to analyze the Golgi structure in prophase. The cells were fixed with MeOH and stained for TGN46 ( gray or green ) and DNA ( red ). B , representative z-stack images are shown, and Golgi objects based on TGN46 staining are numbered (see “ ”). Scale bar, 10 μm. C , the number of Golgi object within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 40). Statistical analysis was performed using Welch’s ANOVA ( F = 319, p = 0.000), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). D and E , The cells were cultured for a further 9.5 h or 10.5 h in siControl or siLAT1 cells, respectively, to analyze Aurora A recruitment in prometaphase cells. Then, the cells were fixed with MeOH and stained for Aurora A ( green ), phospho-Aurora A (pT288) ( red ), and DNA ( cyan ). D , representative z-stack images are shown. Scale bar, 10 μm. E , the fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured (see “ ”), and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Statistical analysis was performed using Welch’s ANOVA ( F = 99.8, p = 0.000 in the left panel ; F = 88.6, p = 0.000 in the right panel ), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). F and G , HeLa S3 cells were treated with DMSO or 30 μM SP600125 for 2 h. F , the cells were fixed with MeOH and stained for TGN46 and DNA. The number of Golgi objects within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). G , the cells were fixed with MeOH and stained for Aurora A, phospho-Aurora A (pT288), and DNA. The fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured, and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Asterisks indicate significant differences [Student’s t test in panel ( F and G ), ∗∗ p < 0.01; ∗∗∗ p < 0.001].

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Staining, Transfection, Control, Cell Culture, Fluorescence

Journal: EJNMMI Research

Article Title: In vitro evaluation of ( S )-2-amino-3-[3-(2- 18 F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid ( 18 F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

doi: 10.1186/s13550-023-00988-1

Figure Lengend Snippet: Properties of tumor-related amino acid transporters

Article Snippet: Rabbit anti-human LAT1 polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ATB 0,+ polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc.) was diluted at 1:50, and rat anti-human xCT monoclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan) was diluted at 1:100.

Techniques:

Journal: EJNMMI Research

Article Title: In vitro evaluation of ( S )-2-amino-3-[3-(2- 18 F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid ( 18 F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

doi: 10.1186/s13550-023-00988-1

Figure Lengend Snippet: Overexpression of human amino acid transporters. Western blot analyses were performed using anti-human A LAT1, B ATB 0,+ , C ASCT2, and D xCT antibodies on membrane protein extracts from the positive-control cells, negative-control cells (CHO-K1 cells), control vector-transfected cells (Mock), expression vector-transfected cells, and gene-specific siRNA-transfected cells (upper panel). The positive-control cells were A MCF-7, B T3M-4, C NCI-H460, and D A549 cells. The same blots were also probed with anti-CD98 antibody (middle panel), and anti-Na + /K + -ATPase antibody as a loading control (lower panel).

Article Snippet: Rabbit anti-human LAT1 polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ATB 0,+ polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc.) was diluted at 1:50, and rat anti-human xCT monoclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan) was diluted at 1:100.

Techniques: Over Expression, Western Blot, Positive Control, Negative Control, Plasmid Preparation, Transfection, Expressing

Journal: EJNMMI Research

Article Title: In vitro evaluation of ( S )-2-amino-3-[3-(2- 18 F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid ( 18 F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

doi: 10.1186/s13550-023-00988-1

Figure Lengend Snippet: Immunofluorescent analysis of human amino acid transporters. Immunofluorescent analyses were performed using anti-human A LAT1, B ATB 0,+ , C ASCT2, and D xCT antibodies (green) on the positive-control cells, negative-control cells (CHO-K1 cells), control vector-transfected cells (Mock), expression vector-transfected cells, and gene-specific siRNA-transfected cells. The positive-control cells were A MCF-7, B T3M-4, C NCI-H460, and D A549 cells. Nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining (blue)

Article Snippet: Rabbit anti-human LAT1 polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ATB 0,+ polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc.) was diluted at 1:50, and rat anti-human xCT monoclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan) was diluted at 1:100.

Techniques: Positive Control, Negative Control, Plasmid Preparation, Transfection, Expressing, Staining

Journal: PLoS ONE

Article Title: Striatal Astrocytes Act as a Reservoir for L-DOPA

doi: 10.1371/journal.pone.0106362

Figure Lengend Snippet: Detection of mRNA expression of DAT (A), protein expression of DAT (B), LAT1 (C), 4F2hc (D), and AADC (E) in primary cultured neurons (N) and astrocytes (G) from mesencephalon (MC) and striatum (Str) of rat embryos using RT-PCR assay (A) and western blot analysis (B–E).

Article Snippet: The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% non-fat milk powder at room temperature for 1 h. Then the blots were incubated with goat polyclonal anti-DAT antibody (1∶200, Santa Cruz Biotechnology, Santa Cruz, CA, #K-20), rabbit polyclonal anti-L-type amino acid transporter LAT1 antibody (dilution; 1∶100, Serotec, Oxford, UK, #AHP735), mouse monoclonal anti-4F2hc antibody (dilution; 1∶200, BD Transduction Laboratories, #611516) or rabbit polyclonal anti-aromatic L-amino acid decarboxylase (AADC) antibody (dilution; 1∶500, Protos Biotech Corporation, New York, NY, #CA201 bDCrab) at room temperature for 1 h. After washing with TBS-T (2×5 min), the blots were reacted with donkey anti-goat IgG (Millipore), donkey anti-rabbit IgG (Amersham Biosciences) or donkey anti-mouse IgG (Millipore) secondary antibody conjugated with horseradish peroxidase (dilution; 1∶2,000 or 5,000) at RT for 1 h. Specific signals of proteins were visualized by chemiluminescence using the ECL Western blotting detection system (Amersham Biosciences).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Molecular Oncology

Article Title: Oncogenic KRAS mutations enhance amino acid uptake by colorectal cancer cells via the hippo signaling effector YAP1

doi: 10.1002/1878-0261.12999

Figure Lengend Snippet: Glutamine and leucine uptake in KRAS mutant CRC cells associated with the expression of selected AATs: (A, B) l ‐glutamine and l ‐leucine uptake levels in KRAS mutant or wt CRC cell lines at 20 min, determined by radiolabeled amino acid transport assay. Data are presented as the mean ± SEM from three independent experiments ( n = 3). Statistical comparisons were computed by one‐way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001). (C) Heat map shows mRNA expression levels (fold change) of SLC‐amino acid transporter genes in different CRC cell lines from three independent biological replicates ( n = 3). (D) Western blot images show the protein expression of AATs (SLC38A2, SLC7A5, and SLC1A5), total‐MEK1/2, phospho‐MEK1/2, total ribosomal S6, and phospho‐ribosomal S6 protein in different CRC cell lines. β‐actin was used as protein loading control. All the experiments were performed three times independently, and the blots shown are representative of three independent replicates.

Article Snippet: Polyclonal rabbit anti‐LAT1/SLC7A5 antibody was provided by TransGenic Inc. (Kumamoto, Japan).

Techniques: Mutagenesis, Expressing, Transport Assay, Western Blot

Journal: Molecular Oncology

Article Title: Oncogenic KRAS mutations enhance amino acid uptake by colorectal cancer cells via the hippo signaling effector YAP1

doi: 10.1002/1878-0261.12999

Figure Lengend Snippet: Knockdown of oncogenic KRAS inhibits the expression of AATs, AA uptake, and mTOR activation in CRC cells. (A) Western blot images show the impact of knockdown of oncogenic KRAS on the protein expression of AATs (SLC7A5/LAT1, SLC1A5/ASCT2, and SLC38A2/SNAT2), KRAS, pan‐RAS, total‐MEK1/2, phospho‐MEK1/2, total‐S6, and phospho‐S6 ribosomal protein in CRC cell lines, SW480, SW620, and HCT116. β‐actin was used as protein loading control. All the experiments were performed three times independently, and the blots shown are representative of three independent replicates. (B, C) Impact of knockdown of KRAS on the l ‐glutamine and l ‐leucine uptake, measured by radioisotope‐based membrane transport assays. Data are presented as the mean ± SEM from three independent experiments ( n = 3). Statistical comparisons were computed by one‐way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Polyclonal rabbit anti‐LAT1/SLC7A5 antibody was provided by TransGenic Inc. (Kumamoto, Japan).

Techniques: Expressing, Activation Assay, Western Blot

Journal: Molecular Oncology

Article Title: Oncogenic KRAS mutations enhance amino acid uptake by colorectal cancer cells via the hippo signaling effector YAP1

doi: 10.1002/1878-0261.12999

Figure Lengend Snippet: Overexpression of mutant KRAS induce the expression of AATs, AA uptake, and mTOR activation in KRAS wt cell line HKe3. (A) Western blot images show the expression of AATs (SLC7A5/LAT1, SLC1A5/ASCT2, and SLC38A2/SNAT2), phospho‐MEK1/2, phospho‐S6 ribosomal protein, and KRAS in HCT116‐KRAS WT/G13D , Hke3‐KRAS WT/G13D− , Hke3‐KRAS WT/WT+ , and Hke3‐KRAS WT/G13D+ cells. KRAS blot show two bands in the lane 3 and 4, as the Hke3‐KRAS WT/WT+ and Hke3‐KRAS WT / G13D+ cells express HA‐tagged and untagged KRAS. All the experiments were performed three times independently, and the blots shown are representative of three independent replicates. (B, C) l ‐glutamine and l ‐leucine uptake levels in HCT116 and HKe3 cell lines expressing KRAS wt and KRAS mutation. (D) Proliferation rate of HCT116‐KRAS WT/G13D , Hke3‐KRAS WT/G13D− , Hke3‐KRAS WT/WT+ , and Hke3‐KRAS WT/G13D+ cells at 24, 48 and 96 h. (E) Bar graph show colony‐forming efficiency of HCT116‐KRAS WT/G13D , Hke3‐KRAS WT/G13D− , Hke3‐KRAS WT/WT+ , and Hke3‐KRAS WT/G13D+ cells. Data are presented as the mean ± SEM from three independent experiments ( n = 3). Statistical comparisons were computed by one‐way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Polyclonal rabbit anti‐LAT1/SLC7A5 antibody was provided by TransGenic Inc. (Kumamoto, Japan).

Techniques: Over Expression, Mutagenesis, Expressing, Activation Assay, Western Blot

Journal: Molecular Oncology

Article Title: Oncogenic KRAS mutations enhance amino acid uptake by colorectal cancer cells via the hippo signaling effector YAP1

doi: 10.1002/1878-0261.12999

Figure Lengend Snippet: Knockdown of AATs inhibits AA uptake, mTOR activation, and proliferation in CRC cells: (A, B) Changes in the l ‐glutamine and l ‐leucine uptake levels in different CRC cell lines (SW480, SW620, HCT116, and DLD‐1) following the transfection of shRNAs against SLC7A5, SLC38A2, and SLC1A5. (C) Impact of knockdowns of SLC1A5, SLC7A5, and SLC38A2 genes on the proliferation of CRC cell lines SW480, SW620, HCT116, and DLD‐1 at 24, 48, 72, 96 and 120h. Data are presented as the mean ± SEM from three independent experiments ( n = 3). Statistical comparisons were computed by one‐way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001). (D) Representative western blot images show the changes in the protein expression of AATs, total‐ and phospho‐S6 ribosomal protein in HCT116 and DLD‐1 cells after the shRNA transfection. β‐actin was used as protein loading control. All the experiments were performed three times independently, and the blots are representative of three independent replicates.

Article Snippet: Polyclonal rabbit anti‐LAT1/SLC7A5 antibody was provided by TransGenic Inc. (Kumamoto, Japan).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, shRNA

Journal: Molecular Oncology

Article Title: Oncogenic KRAS mutations enhance amino acid uptake by colorectal cancer cells via the hippo signaling effector YAP1

doi: 10.1002/1878-0261.12999

Figure Lengend Snippet: Upregulation of AATs in CRC patient samples and their association with oncogenic KRAS mutations: (A–C) Box‐whisker plots show mRNA expression levels of AATs (SLC1A5, SLC7A5, and SLC38A2) in TCGA colon adenocarcinoma patient samples ( n = 286) and normal samples ( n = 41). Box‐whisker plots represent the interquartile range, middle line indicates the median, and the whiskers indicate minimum/maximum values. (D–F) Correlation of mRNA expression levels of AATs (SLC1A5, SLC7A5, and SLC38A2) in CRC patient samples harboring KRAS wt ( n = 317) and KRAS mutations ( n = 217), retrieved from gene expression omnibus database (NCBI‐GEO). Error bars represent mean ± SEM; P ‐values were calculated by unpaired t ‐test.

Article Snippet: Polyclonal rabbit anti‐LAT1/SLC7A5 antibody was provided by TransGenic Inc. (Kumamoto, Japan).

Techniques: Whisker Assay, Expressing