phosphorylated ampk (pampk) thr172 antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

doi: 10.1101/2025.05.04.652104

Figure Lengend Snippet: Nitrate-Sialin2 signaling promotes LKB1-mitochondrial recruitment to activate AMPK phosphorylation a, Schematic workflow of phosphoproteomic analysis. b, Top enriched KEGG pathways in HEK293T cells treated with 4 mM nitrate or control vehicle for 4 h listed by the rank of P value based on DAVID analysis. Data represent three biological replicates per condition. c, IF staining images (left) and quantification (right) of pAMPK T172 in control and SLC17A5 knockout (sg SLC17A5 ) NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. d, Quantification of pAMPK T172 in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2. N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 8e. e, Schematic diagram of subcellular compartment-specific biosensor osABKAR. f, Quantification of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 9d. g, h, IF staining images (upper, representative YFP images, lower, representative pseudocolor images of FRET/CFP ratio show the FRET response; g) and quantification (h) of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2. N = 30 cells from representative experiments of three repeats. i, Quantification of FRET/CFP ratio of Lyso-ExRai-ABKAR in control and sg SLC17A5 NRK cells treated with metformin (Met, 10 mM) for 4 h. N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 9h. j, k, PLA of Sialin/Sialin2-LKB1 in NRK (j) or HEK293T (k) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of HEK293T cells in Extended Data Fig. 10c. l, PLA of Sialin/Sialin2-CaMKK2 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 10d. m, Quantification of FRET/CFP ratio of Mito-ABKAR in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 10e. n, o, IF staining images of LKB1 and MitoTracker in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2 (n). Colocalization quantified by Pearson’s correlation coefficient (o). N = 30 cells from representative experiments of three repeats. p, Immunoblot analysis of pAMPK T172 , AMPK, Flag, and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). Representative images of n = 3 independent experiments were shown. q, r, IF staining images (upper, representative YFP images, lower, representative pseudocolor images of FRET/CFP ratio show the FRET response; q) and quantification (r) of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. s, Schematic illustration of nitrate enhances Sialin2-LKB1 interaction, promotes the recruitment of LKB1 to mitochondria, which in turn promotes the phosphorylation and activation of AMPK. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against CTSB (Santa Cruz Biotechnology, clone H-5, #sc- 365558), Tomm20 (Cell Signaling, clone D8T4N, #42406), LAMP1 (Invitrogen, clone LY1C6, #MA1-164), pAMPK T172 (Cell Signaling, clone 40H9, #2535), AMPK (Cell Signaling, clone D5A2, #5831), LKB1 (Santa Cruz Biotechnology, clone G-12, #sc- 374300), CAMKK2 (Cell Signaling, clone 6G9, #50049), Cox IV (Cell Signaling, clone 3E11, #4850), PGC1α (Santa Cruz Biotechnology, clone D-5, #sc-518025), HA- tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), GAPDH (Cell Signaling, clone 14C10, #2118), ACTB (Proteintech, clone 2D4H5, #66009), HSP90 (Cell Signaling, clone C45G5, #4877), and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), pAMPK T172 (Invitrogen, #PA5-37821), LKB1 (Proteintech, #10746), MT-ND5 (Proteintech, #55410), CYTB (Proteintech, #55090), MT-CO2 (Proteintech, #55070), ATP8 (Proteintech, #26723), TFAM (Proteintech, #22586), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Phospho-proteomics, Control, Staining, Knock-Out, Western Blot, Plasmid Preparation, Activation Assay

Journal: Cells

Article Title: Defective Intracellular Insulin/IGF-1 Signaling Elucidates the Link Between Metabolic Defect and Autoimmunity in Vitiligo

doi: 10.3390/cells14080565

Figure Lengend Snippet: Figure 3. An evaluation of IGF-1, Ins, and glucose effects on the energetic state and oxidative stress. (A) Relative mtDNA quantification was performed on the fibroblast (NHFs n = 23 and VHFs n = 19), keratinocyte (NHKs n = 8 and VHKs n = 8), and melanocyte (NHMs n = 9 and VHMs n = 6) samples in triplicate using qPCR by amplification of mtDNA with mtDNA-specific primers and this was normalized against nuDNA amplification. The histograms present mean fold-change over the corresponding starved control ± SD. The decrease in the mtDNA/nuDNA ratio presented in the vitiligo samples is indicative of the decrease in the mtDNA copy number. (B) The intracellular amount of ATP normalized against the protein content was measured to compare the energetic efficiency of the healthy and vitiligo cells. The histogram report means ± SD of three independent experiments for each cell type. (C) The amount of intracellular ROS generated by metabolic activities confirmed intrinsic oxidative stress in the vitiligo cells (NHFs n = 14, VHFs n = 14; NHKs n = 8, VHKs n = 8; NHMs n = 7, VHMs n = 7). (D) The ROS generated by increasing the glucose concentration was measured with DCFA-DA as reported in c (NHFs n = 8, VHFs n = 8; NHKs n = 6, VHKs n = 6; NHMs n = 3, VHMs n = 3). (E) Representative Western blot images and a densitometric quantification of the pAMPK/AMPK and LC3I/II expression ratios confirmed metabolic impairment and catabolism activation (NHFs n = 4, VHFs n = 4; NHKs n = 4, VHKs n = 4; NHMs n = 3, VHMs n = 3). Cofilin was included as protein load control. Note that * indicates p ≤0.05, ** indicates p ≤0.01, *** indicates p ≤0.001, and **** indicates p ≤0.0001.

Article Snippet: The membranes were then treated with the following primary antibodies: rabbit pAKT-Ser473, pmTOR-Ser2421, mTOR, pS6-Ser235/236, pSer612-IRS1, pAMPK-Thr172, AMPK, LC3, pStat1-Y701 and Stat1 (Cell Signaling Technology, Leiden, The Netherlands), Glu4 (Abcam, Cambridge, UK) and mouse S6, AKT (Cell Signaling Technology, Danvers, MA, USA), and cofilin (BioRad, Hercules, CA, USA) antibodies.

Techniques: Amplification, Control, Generated, Concentration Assay, Western Blot, Expressing, Activation Assay