Journal: Kidney international

Article Title: Over-expression of renal LAT1 and LAT2 and enhanced L-DOPA uptake in SHR immortalized renal proximal tubular cells.

doi: 10.1111/j.1523-1755.2004.00722.x

Figure Lengend Snippet: Fig. 9. Detection of (A) LAT1 and (B) LAT2 in total RNA extracted from WKY and SHR cells using LAT1 and LAT2 rat specific primers.

Article Snippet: Blots were then incubated with rabbit anti-LAT1 polyclonal antibody (1:500; Serotec, Oxford, UK) and rabbit antimouse LAT2 polyclonal antibody [18] (1:2000; kindly provided by Prof. François Verrey) in 5% nonfat dry milk in TBST for 1.5 hours at room temperature.

Techniques:

Journal: Kidney international

Article Title: Over-expression of renal LAT1 and LAT2 and enhanced L-DOPA uptake in SHR immortalized renal proximal tubular cells.

doi: 10.1111/j.1523-1755.2004.00722.x

Figure Lengend Snippet: Fig. 10. Abundance of (A) LAT1 and (B) LAT2 in WKY and SHR cells. Each lane contains equal amount of protein (40 lg). Western blots were repeated 4 to 6 times. Columns indicate relative density and represent the mean of 4 to 6 separate experiments; vertical lines indicate SEM. Significantly different from values for WKY cells. ∗P < 0.05.

Article Snippet: Blots were then incubated with rabbit anti-LAT1 polyclonal antibody (1:500; Serotec, Oxford, UK) and rabbit antimouse LAT2 polyclonal antibody [18] (1:2000; kindly provided by Prof. François Verrey) in 5% nonfat dry milk in TBST for 1.5 hours at room temperature.

Techniques: Western Blot

Journal: Nuclear medicine and biology

Article Title: Enhancing the accumulation level of 3-[ 18 F]fluoro-L-α-methyltyrosine in tumors by preloading probenecid.

doi: 10.1016/j.nucmedbio.2021.11.006

Figure Lengend Snippet: Fig. 5. Analysis of the effect of probenecid on LS180 cell. (A) LAT1 expression and (B) cellular uptake (mean ± SD, n ≥4) of LS180 cell treated with or without probenecid (1 mM). n.s., not significant compared with control group (without probenecid).

Article Snippet: Then, blots were incubated with mouse anti-LAT1 antibody (sc374232, Santa Cruz Biotechnology, Dallas, TX) or rabbit anti-β-actin antibody (#4970, Cell Signaling Technology, Danvers, MA) at 4 °C overnight.

Techniques: Expressing, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

doi: 10.1159/000107537

Figure Lengend Snippet: Fig. 1. Abundance of (A) LAT1 and (B) 4F2hc in WKY and SHR freshly isolated renal proximal tubules and WKY and SHR immortalized renal proximal tubular cells. Columns indicate density and represent the mean of 4-6 separate experiments; vertical lines indicate S.E.M. Significantly different from values for WKY cells (* P<0.05). (C) Each lane contains equal amount of protein (40 µg).

Article Snippet: Blots were then incubated with anti-LAT1 rabbit polyclonal antibody (1:1,500; Serotec, Oxford, UK) or the anti-4F2hc rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the anti β-actin primary antibody (1:10,000; Upstate Biotechnologies) in 5% non-fat dry milk in PBS-T overnight at 4oC.

Techniques: Isolation

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

doi: 10.1159/000107537

Figure Lengend Snippet: Fig. 11. [14C]-L-leucine (0.25 µM) uptake in LAT1 siRNA- and 4F2hc siRNA- treated WKY and SHR immortalized renal proximal tubular cells, for 24h. Columns represent the mean of 4-8 experiments per group; vertical lines show S.E.M.. Significantly different from control value (* P<0.05).

Article Snippet: Blots were then incubated with anti-LAT1 rabbit polyclonal antibody (1:1,500; Serotec, Oxford, UK) or the anti-4F2hc rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the anti β-actin primary antibody (1:10,000; Upstate Biotechnologies) in 5% non-fat dry milk in PBS-T overnight at 4oC.

Techniques: Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

doi: 10.1159/000107537

Figure Lengend Snippet: Fig. 12. Abundance of (A) LAT1 and (B) 4F2hc proteins in LAT1 siRNA- and 4F2hc siRNA-treated WKY and SHR immortalized renal proximal tubular cells, for 24h. Columns represent the mean of 4 experiments per group; vertical lines show S.E.M.. Significantly different from control value (* P<0.05). Each lane contains equal amount of protein (40 µg).

Article Snippet: Blots were then incubated with anti-LAT1 rabbit polyclonal antibody (1:1,500; Serotec, Oxford, UK) or the anti-4F2hc rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the anti β-actin primary antibody (1:10,000; Upstate Biotechnologies) in 5% non-fat dry milk in PBS-T overnight at 4oC.

Techniques: Control

Journal: Scientific Reports

Article Title: DRAM1 enhances the proliferation and metastasis of gastric cancer through the PI3K/AKT/mTOR signaling pathway and energy metabolism

doi: 10.1038/s41598-025-87389-7

Figure Lengend Snippet: DRAM1 mediated energy metabolism in gastric cancer cells through mTOR. ( a ) GO analysis was performed to analyze the differential genes of DRAM1 silencing GC cells and NC GC cells, which were significantly related to energy metabolism pathway. ( b ) KEGG analysis was conducted to examine the differential genes between DRAM1 silenced gastric cancer cells and non-silenced gastric cancer cells. The results revealed a significant association with the energy metabolism pathway. ( c ) KEGG was used to analyze the linkages between the metabolic pathways of differentially enriched genes. ( d ) Knocking down of DRAM1 inhibited the mRNA expression of HK2 (Ctrl vs sh DRAM1 #1,ns; Ctrl vs sh DRAM1 #2, *: P = 0.013), PFK1 (Ctrl vs sh DRAM1 #1, **: P = 0.001; Ctrl vs sh DRAM1 #2, **: P = 0.002), GCK (Ctrl vs sh DRAM1 #1, *: P = 0.011; Ctrl vs sh DRAM1 #2, **: P = 0.004), PKM2 (Ctrl vs sh DRAM1 #1, *: P = 0.042; Ctrl vs sh DRAM1 #2,ns), ATP6V1A (Ctrl vs sh DRAM1 #1, **: P = 0.001; Ctrl vs sh DRAM1 #2, **: P = 0.002), LDHA (Ctrl vs sh DRAM1 #1, **: P = 0.004; Ctrl vs sh DRAM1 #2, **: P = 0.006), c-MYC (Ctrl vs sh DRAM1 #1, ***: P < 0.001; Ctrl vs sh DRAM1 #2, **: P = 0.002) and IDH1 (Ctrl vs sh DRAM1 #1, * : P = 0.038; Ctrl vs sh DRAM1 #2, **: P = 0.005) in gastric cancer cells by q-PCR analysis. ( e ) Western blotting confirmed that knockdown of DRAM1 inhibited the expression of PKM2, HK2, GCK, SLC1A5 and LDHA proteins in gastric cancer cells. ( f ) Western blotting confirmed that overexpression of DRAM1 promoted the expression of PKM2, GCK, SLC1A5, LDHA, PFK1 and SLC7A5 proteins in gastric cancer cells. ( g ) Knockdown of DRAM1 inhibit ATP production in gastric cancer cells (Ctrl vs sh DRAM1 #1, **: P = 0.006; Ctrl vs sh DRAM1 #2, **: P = 0.008). ( h ) Knockdown of DRAM1 inhibited lactate production in gastric cancer cells (Ctrl vs sh DRAM1 #1, **: P = 0.002; Ctrl vs sh DRAM1 #2, **: P = 0.002).

Article Snippet: The following primary antibodies were used: β-actin (proteintech; 66009-1-immunoglobin [Ig]; 1:3000), DRAM1 (Abcam; Ab208160; 1:1000), SLC1A5 (proteintech; 20350-1-AP; 1:2000), SLC7A5 (proteintech; 28670-1-AP; 1:2000), mTOR (proteintech; 66888-1-Ig; 1:2000), p-mTOR (proteintech; 67778-1-Ig; 1:2000), P62 (proteintech; 18420-1-AP; 1:2000), p-AKT (proteintech; 66444-1-Ig; 1:2000), E-cadherin (proteintech; 20874-1-AP; 1:2000), N-cadherin (proteintech; 22018-1-AP; 1:2000), Bax (Cell Signaling Technology; 2772S; 1:1000), Bcl-2 (Cell Signaling Technology; 15071S; 1:1000), PI3K (Wanleibio; WL03380; 1:1000), AKT (Cell Signaling Technology; 9272S; 1:1000), HK2 (Abways; AB3194; 1:1000), PFK1 (Abways; CY8047; 1:1000), GCK (Abways; CY8036; 1:1000), LDHA (bioworld; BS6179;1:1000), PKM2 (Cell Signaling Technology; 4053S; 1:1000), p53 (proteintech; 60283-22-Ig; 1:2000),LC3 (proteintech; 14600-1-AP; 1:2000), anti-rabbit antibody horseradish peroxidase (HRP) rabbit (Cell Signaling Technology; 7074S; 1:3000), and anti-mouse antibody HRP mouse (Cell Signaling Technology; 7076S; 1:3000).

Techniques: Expressing, Western Blot, Knockdown, Over Expression

Journal: bioRxiv

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.1101/2020.01.17.910059

Figure Lengend Snippet: (A) Conductance-voltage relationships were determined for indicated combinations of Kv1.1 and Slc7a5 expressed in LM mouse fibroblasts. Cells were stepped between −120 mV and +110 mV in 10 mV increments, and a tail current voltage of −20 mV. Dashed lines indicate previously reported conductance-voltage relationship in Kv1.2 ± Slc7a5 . Fit parameter for Kv1.1 were (with +Slc7a5 in parentheses): V 1/2 = −34.9 ± 0.3 mV (−37.5 +/- 0.2 mV); k = 6.9 ± 0.9 mV (7.3 ± 0.9 mV). (B) Exemplar records illustrating voltage-dependent activation of Kv1.1 ± Slc7a5. Current traces at −30 mV are bolded in black.

Article Snippet: Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (1:500 dilution, KE026; Trans Genic Inc.) and HRP-conjugated goat anti-rabbit antibody (1:15 000 dilution, SH012; Applied Biological Materials).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.1101/2020.01.17.910059

Figure Lengend Snippet: (A,B) Disinhibtion of Kv1.2 (A) or Kv1.1 (B) was tested by delivering repetitive 50 ms depolarizations to +10 mV (every 2 s), with an interpulse holding voltage of −120 mV. When Kv1.2 is expressed alone, currents remain stable during this protocol (A), whereas Kv1.1 exhibits prominent disinhibition (B). (C-F) Cell-by-cell disinhibition measured before and after a hyperpolarizing pulse train to −120 mV is illustrated for indicated combinations of Kv1.1, Kv1.2 and Slc7a5. A prominent difference between Kv1.2 and Kv1.1 is that Kv1.1 exhibits disinhibition without a requirement for overexpression of Slc7a5 by co-transfection. Current density pre and post-train was compared using a paired t-test (* indicates p < 0.05). Kv1.2 (n=11, no statistical difference); Kv1.2 + Slc7a5 (n=16, p=0.001); Kv1.1 (n=11, p=0.004); Kv1.1 + Slc7a5 (n=13, p=0.003).

Article Snippet: Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (1:500 dilution, KE026; Trans Genic Inc.) and HRP-conjugated goat anti-rabbit antibody (1:15 000 dilution, SH012; Applied Biological Materials).

Techniques: Over Expression, Cotransfection

Journal: bioRxiv

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.1101/2020.01.17.910059

Figure Lengend Snippet: (A) Kv1.1[Y379T] channels were expressed in mouse LM fibroblasts with Slc7a5, as indicated. Exemplar traces illustrate the time course of inactivation elicited by depolarization to +70 mV from a holding potential of −100 mV. Prior to this experiment, currents are disinhibited with a holding voltage of −120 mV, as described in . (B) Mean time constant of inactivation for Kv1.1 ± Slc7a5, over a range of voltages. (C) Time constants of inactivation of Kv1.1 co-expressed with Slc7a5 (Kv1.1 τ = 960 ± 50 ms, n=17; Kv1.1 + Slc7a5 τ = 460 ± 60 ms, n=19). Inactivation time constants were compared with a student’s t-test, (* indicates p < 0.001).

Article Snippet: Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (1:500 dilution, KE026; Trans Genic Inc.) and HRP-conjugated goat anti-rabbit antibody (1:15 000 dilution, SH012; Applied Biological Materials).

Techniques:

Journal: bioRxiv

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.1101/2020.01.17.910059

Figure Lengend Snippet: (A,B) Multiple cell lines expressing different shRNA constructs were transfected with Kv1.1 cDNA and tested for current disinhibition and current magnitude as described in . (C,D) Multiple clonal cell lines were generated by serial dilution of the ShR1 and ShR4 cell lines, and screened for disinhibition of current (C) and Slc7a5 expression by Western blot (D). Further characterization of the ShR4-1 cell line is described in the main text.

Article Snippet: Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (1:500 dilution, KE026; Trans Genic Inc.) and HRP-conjugated goat anti-rabbit antibody (1:15 000 dilution, SH012; Applied Biological Materials).

Techniques: Expressing, shRNA, Construct, Transfection, Generated, Serial Dilution, Western Blot

Journal: bioRxiv

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.1101/2020.01.17.910059

Figure Lengend Snippet: (A) Quantitative real-time PCR was conducted using RNA from parental mouse LM fibroblasts or a stable Slc7a5 shRNA knockdown mouse LM fibroblast cell line (ShR4-1) (n=4, statistical comparison with paired t-test). (B) Western blot of endogenous Slc7a5 in parental LM cells or Slc7a5 shRNA knockdown LM cells (ShR4-1). Actin was used as a loading control. (C) Densitometry measurements of Slc7a5 expression from parental and ShR4-1 LM cells (statistical comparison with paired t-test). Numbers in parentheses above the bars indicate the number of cells examined. (D,E) Exemplar current records illustrating disinhibition of Kv1.1 in response to a −120 mV hyperpolarizing voltage was measured as described in , using the stable Slc7a5 shRNA knockdown (ShR4-1) mouse LM fibroblast cell line. In panel (E), Slc7a5 expression has been rescued by overexpression with a plasmid encoding human Slc7a5, which has 2 mismatches to the shRNA target sequence in the stable line. (F) Disinhibition from a −120 mV pulse train of Kv1.1 on a cell by cell basis in the parental LM cells or ShR4-1 cell line before or after Slc7a5 rescue (n = 9-15). (G) Bar graph depicting the fold disinhibition between the first and the last pulses of a −120 mV pulse train of Kv1.1 in parental LM cells (mean +/- S.D.; 2.96 ± 1.29) or ShR4-1 cells before (1.36 ± 0.20) or after (7.83 ± 4.12) Slc7a5 rescue (n = 9-15, Kruskal-Wallis multiple comparisons test, Dunn’s post-hoc test).

Article Snippet: Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (1:500 dilution, KE026; Trans Genic Inc.) and HRP-conjugated goat anti-rabbit antibody (1:15 000 dilution, SH012; Applied Biological Materials).

Techniques: Real-time Polymerase Chain Reaction, shRNA, Western Blot, Expressing, Over Expression, Plasmid Preparation, Sequencing

Journal: bioRxiv

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.1101/2020.01.17.910059

Figure Lengend Snippet: (A) Cartoons illustrate chimeric channel design, in which increasing segments of Kv1.1 (white) were introduced into Kv1.2 (grey), beginning with the N-terminus. Current disinhibition by a hyperpolarizing train to −120 mV was assessed as described in , in the presence or absence of Slc7a5. (B-E) Conductance-voltage relationships were measured for all chimeric channels, in the presence and absence of Slc7a5. Gating parameters (+Slc7a5 in parentheses) for Kv1.1N/Kv1.2 were: V 1/2 = −8.7 ± 2 mV (−61 ± 3 mV); k = 7 ± 1 mV (8.3 ± 0.5 mV); for Kv1.1S1/Kv1.2: V 1/2 = −21.5 ± 0.7 mV (−42 ± 6 mV); k = 7.1 ± 0.2 mV (9 ± 1 mV), for Kv1.1S2/Kv1.2: V 1/2 = −30.1 ± 0.4 mV (−53 ± 8 mV); k = 7.4 ± 0.2 mV (8.2 ± 0.4 mV), and for Kv1.1S5/Kv1.2: V 1/2 = −25 ± 2 mV (−30 ± 2 mV); k = 7.4 ± 0.3 mV (7.2 ± 0.3 mV). Prominent shifts in voltage-dependent gating were observed in all chimeras except the Kv1.1S5/Kv1.2, comprising primarily the transmembrane domains of Kv1.1.

Article Snippet: Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (1:500 dilution, KE026; Trans Genic Inc.) and HRP-conjugated goat anti-rabbit antibody (1:15 000 dilution, SH012; Applied Biological Materials).

Techniques:

Journal: bioRxiv

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.1101/2020.01.17.910059

Figure Lengend Snippet: (A,C) Top: Chimera design is shown with the voltage-sensing domains of Kv1.1(white) and Kv1.2 (grey) switched as indicated. Gating parameters for the resulting chimeras were (+Slc7a5 in parentheses), for Kv1.1(Kv1.2VSD): V 1/2 = −19.7 ± 2 mV (−62 ± 2 mV); k = 7.5 ± 0.5 mV (7.7 ± 0.9 mV); for Kv1.2(Kv1.1VSD): V 1/2 = −37 ± 2 mV (−53 ± 3 mV); k = 5.5 ± 0.4 mV (7.6 ± 0.9 mV). (B,D) Disinhibition of both chimeras was measured in the presence and absence of Slc7a5, in response to a −120 mV hyperpolarizing train, as described in . Current densities pre- and post-train were compared using a paired t-test (* indicates p < 0.05). Fold disinhibition of Kv1.1(Kv1.2VSD) was 1 ± 0.1 (n=9, no statistical difference), and with Slc7a5 was 8 ± 3 (n=7, p=0.034). Fold disinhibition of Kv1.2(Kv1.1VSD) was 3.9 ± 0.6 (n=7, p=0.016), and with Slc7a5 was 4.3 ± 0.9 (n=11, p=0.001).

Article Snippet: Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (1:500 dilution, KE026; Trans Genic Inc.) and HRP-conjugated goat anti-rabbit antibody (1:15 000 dilution, SH012; Applied Biological Materials).

Techniques:

Journal: Nature Communications

Article Title: Intrinsic resistance to RAS inhibitors is driven by dysregulation of KRAS degradation

doi: 10.1038/s41467-025-67109-5

Figure Lengend Snippet: A , B Heatmaps show the proteomic profiles of proteins in proximity to KRAS, which served as the bait in proximity labeling experiments , . C Immunoblot analysis of the indicated proteins in H727 cells expressing the indicated shRNAs. SLC3A2 (or SLC7A5), p70 S6K p-T389, and Vinculin were detected on the same gel, but different gels for total p70 S6K that was processed in parallel. The immunoblot quantification presented as mean ± SEM; n = 4 technical replicates per group. D VEGFA mRNA levels measured using qPCR in the supernatant of the indicated H727 cells. Data are shown as mean ± SEM, n = 4 technical replicates per group. E ELISA-based VEGFA levels in the supernatant of the indicated H727 cells. Data are shown as mean ± SEM, n = 4 technical replicates per group. F Immunoblotting analysis of SLC7A5 and SCL3A2 expression in DMSO- or RMC7977 (10 nM, 24 hours)-treated H727 cells expressing sh GFP or sh LZTR1 . SLC3A2, SLC7A5, and Vinculin were detected on the same gel. G Immunoblotting analysis of MTOR, SLC7A5 and SCL3A2 expression after lysosome enrichment using HA-immunoprecipitation (Lysosome-IP) in H727 cells expressing TMEM192-3×HA as well as sh GFP or sh LZTR1 . Normalization of the Lysosome fraction using LAMP1 and lysosome wash-out using Vinculin. mTOR, SLC3A2, SLC7A5 and Vinculin (or LAMP1) were detected on the same gel. H Immunofluorescence analysis of H727 cells expressing sh GFP or sh LZTR1 after DMSO- or RMC7977 (10 nM, 24 h)-treated H727 cells using the indicated antibodies. Scale bar 20 µm. Colocalization score for all three proteins is shown as mean ± SEM; n = 4 technical replicates. I PLA was performed on H727 cells expressing sh GFP or sh LZTR1 using antibodies against mTOR and SLC3A2. The number of PLA puncta, indicative of mTOR/SLC3A2 proximity, was quantified per nucleus. Scale bar 30 µm. Data are shown as mean ± SEM, n = 4 technical replicates per group. J PLA was performed on lung tumor sections 10 weeks after post-Cre injection using antibodies against mTOR and SLC3A2. Scale bar 50 µm. The number of PLA puncta, indicative of mTOR/SLC3A2 proximity, was quantified per nucleus. Data are shown as mean ± SEM, n = 4 mice per group, with p -value calculated by two-sided Mann-Whitney test.

Article Snippet: The combinations of antibodies used for PLA on human cells are listed below: On the tumor tissue, a different combination was used: Goat Minus probe (DUO92006) with anti-LAMP1 (Novus Biologicals, AF4320) or SLC3A2 /LAT1 Antibody (Novus Biologicals, NBP1-51935, combined with Rabbit Plus probe (DUO92005) with mTOR (Cell Signaling, #2983).

Techniques: Labeling, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Immunofluorescence, Injection, MANN-WHITNEY