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, IF staining images of pEGFR T1068 with EEA1 in sg SLC17A5 HeLa (a) or NRK (b) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and 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 pEGFR T1068 colocalization with TFR1 by Pearson’s correlation coefficient in sg SLC17A5 HeLa (c) or NRK (d) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). 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