phosphorylated  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Chronic administration of the hydrogen sulfide prodrug SG1002 partially protects against erectile dysfunction resulting from long-term androgen deprivation

doi: 10.1101/2025.05.26.656242

Figure Lengend Snippet: The expression of proteins related to autophagy in mouse corpus cavernosum measured by western blot. Mice underwent sham surgery (Sham) or were surgically castrated (Cast). Two groups of castrated mice were treated with low-dose (CLS) or high-dose (CHS) SG1002. Proteins include light chain 3B (LC3-II), P-p62, p62, and autophagy-related protein (ATG7). Protein expression was normalized to GAPDH or representative controls. Values represent means ± SEM for n = 12 animals per group. * p < 0.05 compared to Sham.

Article Snippet: Primary antibodies were obtained from Cell Signaling Technologies (CST; Danvers, MA, USA), or Protein Tech (PT; Rosemount, IL, USA) and used at the following dilutions: glutamate-cysteine ligase (Gclc, PT #12601-1-AP, 1:1000), optic atrophy type 1 (Opa1, CST #80471, 1:1000), peroxiredoxin 3 (Prdx3, PT #10664-1-AP, 1:1000), peroxiredoxin 5 (Prdx5, PT #17724-1-AP, 1:1000), mitofusin 1 (Mfn1, PT #13798-1-AP, 1:1000), mitofusin 2 (Mfn2, PT #12186-1-AP, 1:1000), NAD(P)H dehydrogenase quinone 1 (Nqo1, CST #62262, 1:1000), thioredoxin 1 (Trx1, CST #2298, 1:1000), thioredoxin-interacting protein (Txnip, PT #18243-1-AP, 1:1000), thioredoxin 2 (Trx2, CST #14907, 1:1000), dynamin-related protein 1 (Drp1, CST #8570, 1:1000), superoxide dismutase 1 (Sod1, PT #10269-1-AP, 1:1000), superoxide dismutase 2 (Sod2, CST #13141, 1:2000), superoxide dismutase 3 (Sod3, Santa Cruz Biotechnology #sc-271170, 1:1000) autophagy-related protein (Atg7, CST #8558, 1:1000), mitochondrial fission 1 (Fis1, PT #10956-1-AP, 1:1000), phosphorylated (Ser 403 ) Sequestosome-1 (P-Sqstm1/p62, CST #39786, 1:1000), sequestosome-1 (Sqstm1/p62, CST #39749, 1:1000), heme oxygenase 1 (HO-1, PT #10701-1-AP, 1:1000), light chain 3B (LC3B, CST #2775, 1:1000), GAPDH (PT #60004-1-Ig, 1:4000).

Techniques: Expressing, Western Blot

Journal: Nature communications

Article Title: TRIM23 mediates cGAS-induced autophagy in anti-HSV defense.

doi: 10.1038/s41467-025-59338-5

Figure Lengend Snippet: Fig. 7 | HSE patient fibroblasts carrying a TBK1 ‘null’ mutation exhibit impaired TRIM23 activation. a Phosphorylation of endogenous p62 (at S403) and TBK1 (at S172) in the WCLs of either healthy control (HC) or patient fibroblasts carrying the heterozygous TBK1 mutation G159A (TBK1(p.G159A/WT)) that were either mock- treated (–) or infected with mutHSV1 (MOI 1) for 16 h, determined by IB using the indicated antibodies. b S39 phosphorylation and ubiquitination of TRIM23-FLAG in transiently transfected TBK1 KO HEK293T cells that were reconstituted with HA- tagged TBK1 WT or mutants, determined at 48 h post-transfection by IP with anti- FLAG and IB with the indicated antibodies. c Endogenous LC3B-I-to-II conversion and p62 phosphorylation (at S403) in TBK1 KO HEK293T cells that were transiently transfected with HA-tagged TBK1 WT or mutants, determined at 24 h post- transfection in the WCLs by IB with the indicated antibodies. d GFP-LC3B puncta formation in HC or patient fibroblasts that were transiently transfected with GFP- LC3B (green) together with either empty vector or the indicated FLAG-tagged

Article Snippet: Primary antibodies thatwere used for IB and theirs dilutions include: antiFLAG (1:2,000, F1804; MilliporeSigma), anti-myc (1:2,000, 2276S; Cell Signaling Technology), anti-HA (1:2,000, H9658;MilliporeSigma), antiβ-actin (1:5,000, A5441; MilliporeSigma), anti-p-S39-TRIM23 (custom made by GenScript, 1:500), anti-TBK1 (1:1,000, 3504S; Cell Signaling Technology), anti-p-S172-TBK1 (1:1,000, 5483S; Cell Signaling Technology), anti-LC3B (1:2,000, CAC-CTB-LC3-1-50; Cosmo Bio), anti-p62 (1:2,000, GP62-C; Progen), anti-p-S403-p62 (1:1,000, 39786; Cell Signaling Technology); anti-IFIT1 (1:1,000, 14769; Cell Signaling Technology), anti-viperin (1:1,000, 13996S; Cell Signaling Technology), antiISG15 (1:1000, sc-166755; Santa Cruz); anti-ubiquitin (1:1,000, sc-8017; Santa Cruz), and anti-HSV-1 (1:1,000, B0114; Dako).

Techniques: Mutagenesis, Activation Assay, Phospho-proteomics, Control, Infection, Ubiquitin Proteomics, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Autophagy-Dependent Regulation of YAP1 by STK38 Governs Recruitment of Differentiated Cells as Progenitor Cells During Regeneration

doi: 10.1101/2025.04.14.648819

Figure Lengend Snippet: (A) Western blot showing active-YAP1, p62, p-p62T269/272, p-p62-S351, p-p62-S405, and β-actin loading control in whole corpus protein extracts from C57BL/6J mice at various injury time points. (B) Confocal, z-stack immunofluorescence showing STK38 is often found within LC3 vesicles in chief cells in the early stage of paligenosis. Scale bar, 5 µm (C,D) Immunoprecipitation assays demonstrate that overexpressed, myc- and mCherry-GFP-tagged STK38 and LC3 can be co-immunoprecipitated in 293-T cells. “Input” = Total protein lysates of 293-T cells co-transfected with empty vector and mCherry-GFP-LC3 plasmid, or myc-STK38 and mCherry-GFP-LC3 plasmids(C), or empty vector and myc-STK38 plasmid, or myc-STK38 and mCherry-GFP-LC3 plasmids(D). “S.E.” and “L.E.” = short and long exposure. Asterisk denotes molecular mass of tagged STK38, which can be distinguished from immunoglobulin heavy chain seen in input. (E) Western blot demonstrates that overexpression of UMF1 in AGS human gastric cell line is sufficient to protect STK38 from degradation induced by rapamycin treatment. (F) Pairwise gene expression correlation analysis using TCGA normal stomach dataset and GTEx stomach dataset reveals a moderate positive linear correlation between UFM1 and STK38 expression. (p=8.9e-16, R=0.52, generated by http://gepia.cancer-pku.cn/ ). (G,H) Western blot analysis shows that Rapamycin inhibits mTORC1 activity and reduces STK38 protein levels in human (G) AGS (gastric), (H) Hep3B2.1-7 (liver), and 293-T (kidney) cell lines. (I) Western blot shows that MG132 proteasome inhibitor not only does not lead to increased STK38 but results in decrease STK38 protein abundance in a dose-dependent manner in the AGS cell line. (J) IHC of wild-type C57BL/6J stomach tissue demonstrates that rapamycin treatment (60 μg/20 g body weight, euthanized 24hours post-injection) effectively reduces the expression of STK38 in the stomach. (K) Western blot demonstrates that rapamycin treatment effectively reduces STK38 protein expression in vivo , as evidenced by gastric protein lysates obtained from experiments like the one depicted in panel J. (L) IHC of wild-type C57BL/6J stomach tissue showing decreased UFM1 in chief cell paligenosis. Red brackets point out the location of the chief cell zone. Scale bar, 50 µm (M) IHC of wild-type C57BL/6J stomach tissue shows that Lys05 treatment effectively preserves STK38 protein levels in HDTAM-treated stomach tissue during stage 3. Scale bar, 100 µm. (N) Immunofluorescence showing reduced STK38 in chief cells (GIF+ cells) of Mist1 −/− mice compared to control. Scale bar, 10 µm (O) IHC of stomach tissue shows active YAP1 increase in chief cells of Mist1 −/− mice (right) chief cells compared to control (left). Red dashed lines point out the outline of chief cells. Note YAP1-positive parietal cells serve as positive controls for immunostaining. Scale bar, 10 µm.

Article Snippet: Membranes were incubated with 3% bovine serum albumin (BSA) overnight at 4 °C with various primary antibodies: anti-STK38 (1:1,000, Proteintech), anti-STK38L (1:1,000, Proteintech), anti-Phospho-STK38/STK38L (Thr444, Thr442, 1:1,000, Thermo Fisher), anti-active YAP1 (1:1,500, Abcam), anti-Phospho-YAP (Ser127, 1:1,000, CST), anti-Phospho-YAP (Ser397, 1:1,000, CST), anti-YAP (1:2,000, CST), anti-Phospho-SQSTM1/p62 (1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser403, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser349, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Thr269/Ser272, 1:1,000, CST), anti-LATS1 (1:1,000, CST), anti-Phospho-LATS1 (Ser909, 1:1,000, CST), anti-Phospho-LATS1 (Thr1079, 1:1,000, CST), anti-NF2 (1:1,000, CST), anti-MST1 (1:1,000, CST), anti-MST2 (1:1,000, CST), anti-Phospho-MST1 (Thr183)/MST2 (Thr180, 1:1,000, CST), anti-MOB1 (1:1,000, CST), anti-SAV1 (1:1,000, CST), anti-RASSF1 (1:1,000, Invitrogen), anti-LC3B (1:2,000, Novus Biologicals), anti-UFM1 (1:1,000, Abcam), anti-Phospho-S6 Ribosomal Protein (Ser240/244, 1:1,000, CST), and anti-beta Actin (1:1,000, Santa Cruz).

Techniques: Western Blot, Control, Immunofluorescence, Immunoprecipitation, Transfection, Plasmid Preparation, Over Expression, Gene Expression, Expressing, Generated, Activity Assay, Quantitative Proteomics, Injection, In Vivo, Immunostaining

Journal: bioRxiv

Article Title: Autophagy-Dependent Regulation of YAP1 by STK38 Governs Recruitment of Differentiated Cells as Progenitor Cells During Regeneration

doi: 10.1101/2025.04.14.648819

Figure Lengend Snippet: (A) Increased levels of active p62 are observed in the pancreas of Cerulein-treated C57BL/6J mice. (B) Total abundance of canonical Hippo pathway kinases remain unchanged in Atf3 −/− mice during paligenosis, and the decrease seen in STK38 early in paligenosis is less marked with STK38 also persisting throughout paligenosis. (C) Quantification of panel B highlights how STK38 abundance declined more gradually and less markedly in Atf3 −/− mice compared to wild-type mice. (D) IHC staining revealed a decrease in STK38 levels in the pancreas of rapamycin-treated mice. Scale bar, 100 µm. (E,F) IHC staining demonstrated a reduction of UMF1 in Mist1 knockout mouse gastric glands (E) and acinar cells (F). Scale bar, 50 µm. (G,H) IHC staining illustrated a decrease in STK38 levels in the stomach (G) and pancreas (H) of Mist1 knockout mice. Scale bar, 50 µm. (I,J) Immunofluorescence staining shows upregulation of active YAP1 in plasma cells of Mist1 knockout mice compared to controls. The immunofluorescence staining of plasma cells (I) with subsequent statistical analysis confirms finding by (J). Scale bar, 50 µm. **** P < 0.0001 (unpaired t-test). Data are presented as mean ± SEM, derived from 10 low-power fields per condition across three independent experiments.

Article Snippet: Membranes were incubated with 3% bovine serum albumin (BSA) overnight at 4 °C with various primary antibodies: anti-STK38 (1:1,000, Proteintech), anti-STK38L (1:1,000, Proteintech), anti-Phospho-STK38/STK38L (Thr444, Thr442, 1:1,000, Thermo Fisher), anti-active YAP1 (1:1,500, Abcam), anti-Phospho-YAP (Ser127, 1:1,000, CST), anti-Phospho-YAP (Ser397, 1:1,000, CST), anti-YAP (1:2,000, CST), anti-Phospho-SQSTM1/p62 (1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser403, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Ser349, 1:1,000, CST), anti-Phospho-SQSTM1/p62 (Thr269/Ser272, 1:1,000, CST), anti-LATS1 (1:1,000, CST), anti-Phospho-LATS1 (Ser909, 1:1,000, CST), anti-Phospho-LATS1 (Thr1079, 1:1,000, CST), anti-NF2 (1:1,000, CST), anti-MST1 (1:1,000, CST), anti-MST2 (1:1,000, CST), anti-Phospho-MST1 (Thr183)/MST2 (Thr180, 1:1,000, CST), anti-MOB1 (1:1,000, CST), anti-SAV1 (1:1,000, CST), anti-RASSF1 (1:1,000, Invitrogen), anti-LC3B (1:2,000, Novus Biologicals), anti-UFM1 (1:1,000, Abcam), anti-Phospho-S6 Ribosomal Protein (Ser240/244, 1:1,000, CST), and anti-beta Actin (1:1,000, Santa Cruz).

Techniques: Immunohistochemistry, Knock-Out, Immunofluorescence, Staining, Clinical Proteomics, Derivative Assay