Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Schematic diagram of the phosphoproteomic workflow. b, Top enriched KEGG pathways in HEK293T cells treated with 4 mM nitrate or control for 4 h listed by the rank of P value based on DAVID analysis. Data represent three biological replicates per condition. c, d, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, and RAB7 in HEK293T cells treated with nitrate (4 mM, 0, 1, 2, and 4 h). Representative images of n = 3 independent experiments were shown. See full quantitation in . e, f, Immunofluorescence (IF) staining images (left) and quantification (right) of pAKT S473 in HeLa (e) and NRK (f) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. g–j, IF staining images and quantitation of endocytic markers EEA1 (g), RAB5 (h), RAB7 (i), and TFR1 (j) in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k–n, IF quantitation of EEA1 (k, l) and RAB7 (m, n) in HeLa cells treated with EGF (50 ng/ml) or insulin (10 μg/ml) for 5 min. N = 30 cells from representative experiments of three repeats. See images in . o, p, IF staining images of pAKT S473 with EEA1 (o) or TFR1 (p) in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. q, Experimental design for subcellular fractionation of endosomal compartments. r, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in the plasma membrane (PM) and endosomal (End) fractions from HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. s, t, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in HEK293T cells pre- treated with nitrate (4 mM, 4 h) followed by chlorpromazine (CPZ, 6 μg/ml; s) or nystatin (25 μg/ml; t) for 30 min. Representative images of n = 3 independent experiments were shown. See full immunoblot images in . u, Schematic illustration of nitrate-triggered endocytosis and activation of PI3K- AKT signaling on endosomes. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Control, Western Blot, Quantitation Assay, Immunofluorescence, Staining, Fractionation, Clinical Proteomics, Membrane, Activation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a–c, Immunoblot analysis of p110α, RAB5, and RAB7 in HEK293T cells treated with nitrate (4 mM, 0, 1, 2, and 4 h). See full immunoblot images and quantitation in . N = 3. d–g, IF staining images of EEA1 (d, e) and RAB7 (f, g) in HeLa cells treated with EGF (50 ng/ml) or insulin (10 μg/ml) for 5 min. N = 30 cells from representative experiments of three repeats. See quantitation in . h–k, IF staining images and quantitation of RAB5 in HeLa cells treated with EGF (50 ng/ml; h, i) or insulin (10 μg/ml; j, k) for 5 min. N = 30 cells from representative experiments of three repeats. l, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in HEK293T cells pre- treated with nitrate (4 mM, 4 h) followed by nystatin (25 μg/ml) for 30 min. Representative images of n = 3 independent experiments were shown. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Western Blot, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Schematic illustration of the Sialin2-TurboID proximity labeling workflow. b, c, Top enriched GO terms (b) and KEGG pathways (c) for Sialin2-interacting proteins in HEK293T cells, ranked by P value according to DAVID analysis. Data represent three biological replicates per condition. d, e, IF staining images (d) and quantitation (e) of EGFP-Sialin with EEA1, RAB5, or RAB7 in HeLa cells stable expressed EGFP-Sialin. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. f, IF quantitation of Sialin-HA colocalization with EEA1, RAB5, or RAB7 by Pearson’s correlation coefficient in HeLa cells stable expressed Sialin-HA. N = 30 cells from representative experiments of three repeats. See images in . g, h, IF staining images (g) and quantitation (h) of mCherry-Sialin2 with EEA1, RAB5, or RAB7 in HeLa cells stable expressed mCherry-Sialin2. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. i, IF quantitation of Sialin2-Flag colocalization with EEA1, RAB5, or RAB7 by Pearson’s correlation coefficient in HeLa cells stable expressed Sialin2-Flag. N = 30 cells from representative experiments of three repeats. See images in . j, k, IF staining images (j) and quantitation (k) of Sialin with RAB7 in HeLa cells treated with EGF (50 ng/ml) for 0, 5, and 15 min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. l, IF quantitation of Sialin colocalization with RAB7 by Pearson’s correlation coefficient in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15 min. N = 30 cells from representative experiments of three repeats. See images in . m, n, IF staining images (m) and quantitation (n) of Sialin2 with RAB7 in HeLa cells treated with EGF (50 ng/ml) for 0, 5, and 15 min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. o, IF quantitation of Sialin2 colocalization with RAB7 by Pearson’s correlation coefficient in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15 min. N = 30 cells from representative experiments of three repeats. See images in . p, Immunoblot analysis of Sialin and Sialin2 in PM and End fractions from HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. q, Co-IP of Sialin2 with RAB5, RAB7, TFR1, or AKT in HEK293T cells treated with nitrate (4 mM, 4 h). Representative data of n = 3 independent experiments were shown. r, s, IF staining images and quantitation of Sialin (r) or Sialin2 (s) with RAB7 in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. t–w, IF quantitation of Sialin/Sialin2 colocalization with RAB5 (t, u) or TFR1 (v, w) by Pearson’s correlation coefficient in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats.See RAB5 images in and TFR1 images in . x, Schematic illustration of nitrate uptaking via endocytosis induces CTSB- dependent cleavage of Sialin, generating Sialin2, which accumulates on endosomes. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Labeling, Staining, Quantitation Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, IF staining images of Sialin-HA with EEA1, RAB5, or RAB7 in HeLa cells stable expressed Sialin-HA. N = 30 cells from representative experiments of three repeats. See quantitation in . b, IF staining images of Sialin2-Flag with EEA1, RAB5, or RAB7 in HeLa cells stable expressed Sialin2-Flag. N = 30 cells from representative experiments of three repeats. See quantitation in . c, IF staining images of Sialin with RAB7 in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15min. N = 30 cells from representative experiments of three repeats. See quantitation in . d–g, IF staining images and quantitation of Sialin with EEA1 in HeLa cells treated with EGF (50 ng/ml; d, e) or insulin (10 μg/ml; f, g) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. h–k, IF staining images and quantitation of Sialin with RAB5 in HeLa cells treated with EGF (50 ng/ml; h, i) or insulin (10 μg/ml; j, k) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. l–o, IF staining images and quantitation of Sialin with TFR1 in HeLa cells treated with EGF (50 ng/ml; l, m) or insulin (10 μg/ml; n, o) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, IF staining images and quantitation of Sialin (a) or Sialin2 (b) with EEA1 in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. c, d, IF staining images of Sialin (c) or Sialin2 (d) with RAB5 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . e, f, IF staining images of Sialin (e) or Sialin2 (f) with TFR1 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, Co-IP of RAB5 (a) or RAB7 (b) with Sialin/Sialin2 in HEK293T cells treated with nitrate (4 mM, 4 h). Representative data of n = 3 independent experiments were shown. c, d, Proximity ligation assay (PLA) of Sialin/Sialin2-pAKT T308 in HeLa (c) or NRK (d) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of NRK cells in . e, f, PLA of Sialin/Sialin2-pAKT S473 in HeLa (e) or NRK (f) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of NRK cells in . g, h, IF staining images and quantitation of Lyn colocalization with RAB5 (g) or RAB7 (h) by Pearson’s correlation coefficient in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. i, j, PLA of Sialin/Sialin2-Lyn (i) or Sialin/Sialin2-EGFR (j) in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k, l, PLA of Lyn-EGFR in nitrate-treated (4 mM, 4 h; k) or SLC17A5 knockout (sg SLC17A5 ; l) HeLa cells. N = 30 cells from representative experiments of three repeats. See images in . m, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and sg SLC17A5 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. n, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and Sialin2-overexpressing (OE-Sialin2) HEK293T cells treated with nitrate (4 mM, 4 h). N = 3. See full immunoblot images in . o, p, IF quantitation of pAKT S473 with EEA1 in sg SLC17A5 HeLa (o) or NRK (p) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with EGF (50 ng/ml, 5 min). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images in . q, r, IF staining images and quantitation of pAKT S473 with EEA1 in sg SLC17A5 HeLa (q) or NRK (r) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images of NRK cells in . s, t, IF quantitation of pEGFR T1068 with EEA1 in sg SLC17A5 HeLa (s) or NRK (t) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images in . u, Schematic illustration of nitrate-induced endocytosis enriches Sialin2 on endosomes, where it recruits LYN kinase to phosphorylate EGFR and activate PI3K- AKT signaling specifically within endosomal compartments. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Co-Immunoprecipitation Assay, Proximity Ligation Assay, Staining, Quantitation Assay, Knock-Out, Western Blot, Control

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, PLA of Sialin/Sialin2-pAKT T308 (a) or Sialin/Sialin2-pAKT S473 (b) in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . c–d, IF staining images and quantitation of Lyn colocalization with RAB5 (c) or Lyn colocalization with RAB7 (d) by Pearson’s correlation coefficient in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. e, f, PLA of Sialin/Sialin2-Lyn (e) or Sialin/Sialin2-EGFR (f) in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. g, h, PLA of Lyn-EGFR in nitrate-treated (4 mM, 4 h) HeLa (g) or NRK (h) cells. N = 30 cells from representative experiments of three repeats. See quantitation of HeLa cells in . i, j, IF staining images and quantitation of Lyn colocalization with EGFR by Pearson’s correlation coefficient in HeLa (i) and NRK (j) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k, l, PLA of Lyn-EGFR in sg SLC17A5 HeLa (k) or NRK (l) cells. N = 30 cells from representative experiments of three repeats. See quantitation of HeLa cells in . m, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Lyn in control and Lyn knockdown (sh Lyn ) HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Quantitation Assay, Staining, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and sg SLC17A5 HEK293T cells treated with EGF (50 ng/ml, 5 min). Representative images of n = 3 independent experiments were shown. b, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and OE-Sialin2 HEK293T cells treated with EGF (50 ng/ml, 5 min). Representative images of n = 3 independent experiments were shown. c, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and sg SLC17A5 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. d, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and OE-Sialin2 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. e, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and OE-Sialin2 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test.

Article Snippet: For immunoprecipitation, antibody-conjugated agarose was purchased from Santa Cruz Biotechnology, including agarose-conjugated antibodies against anti-RAB5 (#sc-46692AC) and anti-RAB7 (#sc-376362AC).

Techniques: Western Blot, Control, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Schematic diagram of the phosphoproteomic workflow. b, Top enriched KEGG pathways in HEK293T cells treated with 4 mM nitrate or control for 4 h listed by the rank of P value based on DAVID analysis. Data represent three biological replicates per condition. c, d, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, and RAB7 in HEK293T cells treated with nitrate (4 mM, 0, 1, 2, and 4 h). Representative images of n = 3 independent experiments were shown. See full quantitation in . e, f, Immunofluorescence (IF) staining images (left) and quantification (right) of pAKT S473 in HeLa (e) and NRK (f) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. g–j, IF staining images and quantitation of endocytic markers EEA1 (g), RAB5 (h), RAB7 (i), and TFR1 (j) in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k–n, IF quantitation of EEA1 (k, l) and RAB7 (m, n) in HeLa cells treated with EGF (50 ng/ml) or insulin (10 μg/ml) for 5 min. N = 30 cells from representative experiments of three repeats. See images in . o, p, IF staining images of pAKT S473 with EEA1 (o) or TFR1 (p) in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. q, Experimental design for subcellular fractionation of endosomal compartments. r, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in the plasma membrane (PM) and endosomal (End) fractions from HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. s, t, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in HEK293T cells pre- treated with nitrate (4 mM, 4 h) followed by chlorpromazine (CPZ, 6 μg/ml; s) or nystatin (25 μg/ml; t) for 30 min. Representative images of n = 3 independent experiments were shown. See full immunoblot images in . u, Schematic illustration of nitrate-triggered endocytosis and activation of PI3K- AKT signaling on endosomes. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Control, Western Blot, Quantitation Assay, Immunofluorescence, Staining, Fractionation, Clinical Proteomics, Membrane, Activation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a–c, Immunoblot analysis of p110α, RAB5, and RAB7 in HEK293T cells treated with nitrate (4 mM, 0, 1, 2, and 4 h). See full immunoblot images and quantitation in . N = 3. d–g, IF staining images of EEA1 (d, e) and RAB7 (f, g) in HeLa cells treated with EGF (50 ng/ml) or insulin (10 μg/ml) for 5 min. N = 30 cells from representative experiments of three repeats. See quantitation in . h–k, IF staining images and quantitation of RAB5 in HeLa cells treated with EGF (50 ng/ml; h, i) or insulin (10 μg/ml; j, k) for 5 min. N = 30 cells from representative experiments of three repeats. l, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in HEK293T cells pre- treated with nitrate (4 mM, 4 h) followed by nystatin (25 μg/ml) for 30 min. Representative images of n = 3 independent experiments were shown. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Western Blot, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Schematic illustration of the Sialin2-TurboID proximity labeling workflow. b, c, Top enriched GO terms (b) and KEGG pathways (c) for Sialin2-interacting proteins in HEK293T cells, ranked by P value according to DAVID analysis. Data represent three biological replicates per condition. d, e, IF staining images (d) and quantitation (e) of EGFP-Sialin with EEA1, RAB5, or RAB7 in HeLa cells stable expressed EGFP-Sialin. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. f, IF quantitation of Sialin-HA colocalization with EEA1, RAB5, or RAB7 by Pearson’s correlation coefficient in HeLa cells stable expressed Sialin-HA. N = 30 cells from representative experiments of three repeats. See images in . g, h, IF staining images (g) and quantitation (h) of mCherry-Sialin2 with EEA1, RAB5, or RAB7 in HeLa cells stable expressed mCherry-Sialin2. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. i, IF quantitation of Sialin2-Flag colocalization with EEA1, RAB5, or RAB7 by Pearson’s correlation coefficient in HeLa cells stable expressed Sialin2-Flag. N = 30 cells from representative experiments of three repeats. See images in . j, k, IF staining images (j) and quantitation (k) of Sialin with RAB7 in HeLa cells treated with EGF (50 ng/ml) for 0, 5, and 15 min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. l, IF quantitation of Sialin colocalization with RAB7 by Pearson’s correlation coefficient in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15 min. N = 30 cells from representative experiments of three repeats. See images in . m, n, IF staining images (m) and quantitation (n) of Sialin2 with RAB7 in HeLa cells treated with EGF (50 ng/ml) for 0, 5, and 15 min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. o, IF quantitation of Sialin2 colocalization with RAB7 by Pearson’s correlation coefficient in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15 min. N = 30 cells from representative experiments of three repeats. See images in . p, Immunoblot analysis of Sialin and Sialin2 in PM and End fractions from HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. q, Co-IP of Sialin2 with RAB5, RAB7, TFR1, or AKT in HEK293T cells treated with nitrate (4 mM, 4 h). Representative data of n = 3 independent experiments were shown. r, s, IF staining images and quantitation of Sialin (r) or Sialin2 (s) with RAB7 in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. t–w, IF quantitation of Sialin/Sialin2 colocalization with RAB5 (t, u) or TFR1 (v, w) by Pearson’s correlation coefficient in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats.See RAB5 images in and TFR1 images in . x, Schematic illustration of nitrate uptaking via endocytosis induces CTSB- dependent cleavage of Sialin, generating Sialin2, which accumulates on endosomes. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Labeling, Staining, Quantitation Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, IF staining images of Sialin-HA with EEA1, RAB5, or RAB7 in HeLa cells stable expressed Sialin-HA. N = 30 cells from representative experiments of three repeats. See quantitation in . b, IF staining images of Sialin2-Flag with EEA1, RAB5, or RAB7 in HeLa cells stable expressed Sialin2-Flag. N = 30 cells from representative experiments of three repeats. See quantitation in . c, IF staining images of Sialin with RAB7 in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15min. N = 30 cells from representative experiments of three repeats. See quantitation in . d–g, IF staining images and quantitation of Sialin with EEA1 in HeLa cells treated with EGF (50 ng/ml; d, e) or insulin (10 μg/ml; f, g) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. h–k, IF staining images and quantitation of Sialin with RAB5 in HeLa cells treated with EGF (50 ng/ml; h, i) or insulin (10 μg/ml; j, k) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. l–o, IF staining images and quantitation of Sialin with TFR1 in HeLa cells treated with EGF (50 ng/ml; l, m) or insulin (10 μg/ml; n, o) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, IF staining images and quantitation of Sialin (a) or Sialin2 (b) with EEA1 in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. c, d, IF staining images of Sialin (c) or Sialin2 (d) with RAB5 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . e, f, IF staining images of Sialin (e) or Sialin2 (f) with TFR1 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, Co-IP of RAB5 (a) or RAB7 (b) with Sialin/Sialin2 in HEK293T cells treated with nitrate (4 mM, 4 h). Representative data of n = 3 independent experiments were shown. c, d, Proximity ligation assay (PLA) of Sialin/Sialin2-pAKT T308 in HeLa (c) or NRK (d) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of NRK cells in . e, f, PLA of Sialin/Sialin2-pAKT S473 in HeLa (e) or NRK (f) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of NRK cells in . g, h, IF staining images and quantitation of Lyn colocalization with RAB5 (g) or RAB7 (h) by Pearson’s correlation coefficient in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. i, j, PLA of Sialin/Sialin2-Lyn (i) or Sialin/Sialin2-EGFR (j) in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k, l, PLA of Lyn-EGFR in nitrate-treated (4 mM, 4 h; k) or SLC17A5 knockout (sg SLC17A5 ; l) HeLa cells. N = 30 cells from representative experiments of three repeats. See images in . m, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and sg SLC17A5 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. n, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and Sialin2-overexpressing (OE-Sialin2) HEK293T cells treated with nitrate (4 mM, 4 h). N = 3. See full immunoblot images in . o, p, IF quantitation of pAKT S473 with EEA1 in sg SLC17A5 HeLa (o) or NRK (p) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with EGF (50 ng/ml, 5 min). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images in . q, r, IF staining images and quantitation of pAKT S473 with EEA1 in sg SLC17A5 HeLa (q) or NRK (r) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images of NRK cells in . s, t, IF quantitation of pEGFR T1068 with EEA1 in sg SLC17A5 HeLa (s) or NRK (t) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images in . u, Schematic illustration of nitrate-induced endocytosis enriches Sialin2 on endosomes, where it recruits LYN kinase to phosphorylate EGFR and activate PI3K- AKT signaling specifically within endosomal compartments. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Co-Immunoprecipitation Assay, Proximity Ligation Assay, Staining, Quantitation Assay, Knock-Out, Western Blot, Control

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, PLA of Sialin/Sialin2-pAKT T308 (a) or Sialin/Sialin2-pAKT S473 (b) in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . c–d, IF staining images and quantitation of Lyn colocalization with RAB5 (c) or Lyn colocalization with RAB7 (d) by Pearson’s correlation coefficient in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. e, f, PLA of Sialin/Sialin2-Lyn (e) or Sialin/Sialin2-EGFR (f) in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. g, h, PLA of Lyn-EGFR in nitrate-treated (4 mM, 4 h) HeLa (g) or NRK (h) cells. N = 30 cells from representative experiments of three repeats. See quantitation of HeLa cells in . i, j, IF staining images and quantitation of Lyn colocalization with EGFR by Pearson’s correlation coefficient in HeLa (i) and NRK (j) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k, l, PLA of Lyn-EGFR in sg SLC17A5 HeLa (k) or NRK (l) cells. N = 30 cells from representative experiments of three repeats. See quantitation of HeLa cells in . m, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Lyn in control and Lyn knockdown (sh Lyn ) HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Quantitation Assay, Staining, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and sg SLC17A5 HEK293T cells treated with EGF (50 ng/ml, 5 min). Representative images of n = 3 independent experiments were shown. b, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and OE-Sialin2 HEK293T cells treated with EGF (50 ng/ml, 5 min). Representative images of n = 3 independent experiments were shown. c, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and sg SLC17A5 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. d, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and OE-Sialin2 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. e, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and OE-Sialin2 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test.

Article Snippet: HRP-conjugated RAB5 (Santa Cruz Biotechnology, #sc-46692HRP), RAB7 (Santa Cruz Biotechnology, #sc- 376362HRP), and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Western Blot, Control, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Schematic diagram of the phosphoproteomic workflow. b, Top enriched KEGG pathways in HEK293T cells treated with 4 mM nitrate or control for 4 h listed by the rank of P value based on DAVID analysis. Data represent three biological replicates per condition. c, d, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, and RAB7 in HEK293T cells treated with nitrate (4 mM, 0, 1, 2, and 4 h). Representative images of n = 3 independent experiments were shown. See full quantitation in . e, f, Immunofluorescence (IF) staining images (left) and quantification (right) of pAKT S473 in HeLa (e) and NRK (f) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. g–j, IF staining images and quantitation of endocytic markers EEA1 (g), RAB5 (h), RAB7 (i), and TFR1 (j) in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k–n, IF quantitation of EEA1 (k, l) and RAB7 (m, n) in HeLa cells treated with EGF (50 ng/ml) or insulin (10 μg/ml) for 5 min. N = 30 cells from representative experiments of three repeats. See images in . o, p, IF staining images of pAKT S473 with EEA1 (o) or TFR1 (p) in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. q, Experimental design for subcellular fractionation of endosomal compartments. r, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in the plasma membrane (PM) and endosomal (End) fractions from HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. s, t, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in HEK293T cells pre- treated with nitrate (4 mM, 4 h) followed by chlorpromazine (CPZ, 6 μg/ml; s) or nystatin (25 μg/ml; t) for 30 min. Representative images of n = 3 independent experiments were shown. See full immunoblot images in . u, Schematic illustration of nitrate-triggered endocytosis and activation of PI3K- AKT signaling on endosomes. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Control, Western Blot, Quantitation Assay, Immunofluorescence, Staining, Fractionation, Clinical Proteomics, Membrane, Activation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a–c, Immunoblot analysis of p110α, RAB5, and RAB7 in HEK293T cells treated with nitrate (4 mM, 0, 1, 2, and 4 h). See full immunoblot images and quantitation in . N = 3. d–g, IF staining images of EEA1 (d, e) and RAB7 (f, g) in HeLa cells treated with EGF (50 ng/ml) or insulin (10 μg/ml) for 5 min. N = 30 cells from representative experiments of three repeats. See quantitation in . h–k, IF staining images and quantitation of RAB5 in HeLa cells treated with EGF (50 ng/ml; h, i) or insulin (10 μg/ml; j, k) for 5 min. N = 30 cells from representative experiments of three repeats. l, Immunoblot analysis of pAKT T308 , pAKT S473 , and AKT in HEK293T cells pre- treated with nitrate (4 mM, 4 h) followed by nystatin (25 μg/ml) for 30 min. Representative images of n = 3 independent experiments were shown. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Western Blot, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Schematic illustration of the Sialin2-TurboID proximity labeling workflow. b, c, Top enriched GO terms (b) and KEGG pathways (c) for Sialin2-interacting proteins in HEK293T cells, ranked by P value according to DAVID analysis. Data represent three biological replicates per condition. d, e, IF staining images (d) and quantitation (e) of EGFP-Sialin with EEA1, RAB5, or RAB7 in HeLa cells stable expressed EGFP-Sialin. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. f, IF quantitation of Sialin-HA colocalization with EEA1, RAB5, or RAB7 by Pearson’s correlation coefficient in HeLa cells stable expressed Sialin-HA. N = 30 cells from representative experiments of three repeats. See images in . g, h, IF staining images (g) and quantitation (h) of mCherry-Sialin2 with EEA1, RAB5, or RAB7 in HeLa cells stable expressed mCherry-Sialin2. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. i, IF quantitation of Sialin2-Flag colocalization with EEA1, RAB5, or RAB7 by Pearson’s correlation coefficient in HeLa cells stable expressed Sialin2-Flag. N = 30 cells from representative experiments of three repeats. See images in . j, k, IF staining images (j) and quantitation (k) of Sialin with RAB7 in HeLa cells treated with EGF (50 ng/ml) for 0, 5, and 15 min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. l, IF quantitation of Sialin colocalization with RAB7 by Pearson’s correlation coefficient in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15 min. N = 30 cells from representative experiments of three repeats. See images in . m, n, IF staining images (m) and quantitation (n) of Sialin2 with RAB7 in HeLa cells treated with EGF (50 ng/ml) for 0, 5, and 15 min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. o, IF quantitation of Sialin2 colocalization with RAB7 by Pearson’s correlation coefficient in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15 min. N = 30 cells from representative experiments of three repeats. See images in . p, Immunoblot analysis of Sialin and Sialin2 in PM and End fractions from HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. q, Co-IP of Sialin2 with RAB5, RAB7, TFR1, or AKT in HEK293T cells treated with nitrate (4 mM, 4 h). Representative data of n = 3 independent experiments were shown. r, s, IF staining images and quantitation of Sialin (r) or Sialin2 (s) with RAB7 in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. t–w, IF quantitation of Sialin/Sialin2 colocalization with RAB5 (t, u) or TFR1 (v, w) by Pearson’s correlation coefficient in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats.See RAB5 images in and TFR1 images in . x, Schematic illustration of nitrate uptaking via endocytosis induces CTSB- dependent cleavage of Sialin, generating Sialin2, which accumulates on endosomes. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Labeling, Staining, Quantitation Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, IF staining images of Sialin-HA with EEA1, RAB5, or RAB7 in HeLa cells stable expressed Sialin-HA. N = 30 cells from representative experiments of three repeats. See quantitation in . b, IF staining images of Sialin2-Flag with EEA1, RAB5, or RAB7 in HeLa cells stable expressed Sialin2-Flag. N = 30 cells from representative experiments of three repeats. See quantitation in . c, IF staining images of Sialin with RAB7 in HeLa cells treated with insulin (10 μg/ml) for 0, 5, and 15min. N = 30 cells from representative experiments of three repeats. See quantitation in . d–g, IF staining images and quantitation of Sialin with EEA1 in HeLa cells treated with EGF (50 ng/ml; d, e) or insulin (10 μg/ml; f, g) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. h–k, IF staining images and quantitation of Sialin with RAB5 in HeLa cells treated with EGF (50 ng/ml; h, i) or insulin (10 μg/ml; j, k) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. l–o, IF staining images and quantitation of Sialin with TFR1 in HeLa cells treated with EGF (50 ng/ml; l, m) or insulin (10 μg/ml; n, o) for 0, 5, and 15min. Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, IF staining images and quantitation of Sialin (a) or Sialin2 (b) with EEA1 in HeLa cells treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. c, d, IF staining images of Sialin (c) or Sialin2 (d) with RAB5 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . e, f, IF staining images of Sialin (e) or Sialin2 (f) with TFR1 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, Co-IP of RAB5 (a) or RAB7 (b) with Sialin/Sialin2 in HEK293T cells treated with nitrate (4 mM, 4 h). Representative data of n = 3 independent experiments were shown. c, d, Proximity ligation assay (PLA) of Sialin/Sialin2-pAKT T308 in HeLa (c) or NRK (d) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of NRK cells in . e, f, PLA of Sialin/Sialin2-pAKT S473 in HeLa (e) or NRK (f) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of NRK cells in . g, h, IF staining images and quantitation of Lyn colocalization with RAB5 (g) or RAB7 (h) by Pearson’s correlation coefficient in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. i, j, PLA of Sialin/Sialin2-Lyn (i) or Sialin/Sialin2-EGFR (j) in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k, l, PLA of Lyn-EGFR in nitrate-treated (4 mM, 4 h; k) or SLC17A5 knockout (sg SLC17A5 ; l) HeLa cells. N = 30 cells from representative experiments of three repeats. See images in . m, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and sg SLC17A5 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. n, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and Sialin2-overexpressing (OE-Sialin2) HEK293T cells treated with nitrate (4 mM, 4 h). N = 3. See full immunoblot images in . o, p, IF quantitation of pAKT S473 with EEA1 in sg SLC17A5 HeLa (o) or NRK (p) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with EGF (50 ng/ml, 5 min). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images in . q, r, IF staining images and quantitation of pAKT S473 with EEA1 in sg SLC17A5 HeLa (q) or NRK (r) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images of NRK cells in . s, t, IF quantitation of pEGFR T1068 with EEA1 in sg SLC17A5 HeLa (s) or NRK (t) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. See images in . u, Schematic illustration of nitrate-induced endocytosis enriches Sialin2 on endosomes, where it recruits LYN kinase to phosphorylate EGFR and activate PI3K- AKT signaling specifically within endosomal compartments. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Co-Immunoprecipitation Assay, Proximity Ligation Assay, Staining, Quantitation Assay, Knock-Out, Western Blot, Control

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, b, PLA of Sialin/Sialin2-pAKT T308 (a) or Sialin/Sialin2-pAKT S473 (b) in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See quantitation in . c–d, IF staining images and quantitation of Lyn colocalization with RAB5 (c) or Lyn colocalization with RAB7 (d) by Pearson’s correlation coefficient in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. e, f, PLA of Sialin/Sialin2-Lyn (e) or Sialin/Sialin2-EGFR (f) in NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. g, h, PLA of Lyn-EGFR in nitrate-treated (4 mM, 4 h) HeLa (g) or NRK (h) cells. N = 30 cells from representative experiments of three repeats. See quantitation of HeLa cells in . i, j, IF staining images and quantitation of Lyn colocalization with EGFR by Pearson’s correlation coefficient in HeLa (i) and NRK (j) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. k, l, PLA of Lyn-EGFR in sg SLC17A5 HeLa (k) or NRK (l) cells. N = 30 cells from representative experiments of three repeats. See quantitation of HeLa cells in . m, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Lyn in control and Lyn knockdown (sh Lyn ) HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Quantitation Assay, Staining, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and sg SLC17A5 HEK293T cells treated with EGF (50 ng/ml, 5 min). Representative images of n = 3 independent experiments were shown. b, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and OE-Sialin2 HEK293T cells treated with EGF (50 ng/ml, 5 min). Representative images of n = 3 independent experiments were shown. c, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and sg SLC17A5 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. d, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, p110α, RAB5, RAB7, and Sialin2 in control and OE-Sialin2 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. e, Immunoblot analysis of pAKT T308 , pAKT S473 , AKT, and Sialin2 in PM and End fractions from control and OE-Sialin2 HEK293T cells treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Western Blot, Control, Quantitation Assay