Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: Antibodies used for the western blot.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Western Blot, Immunofluorescence, Immunoprecipitation

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: Inhibition of ASK1 and MKK3/6 does not inhibit p38MAPK activation. (a) mRNA levels of ASK1 showed siRNA to ASK1 decreases its expression by 89% (P < .0001) compared to NT siRNA. A two-tailed t-test was used to test statistical significance. (b) AECs treated with CSE induced P-p38MAPK, but treatment with siRNA to ASK1 and CSE did not reduce p38MAPK phosphorylation. (c) mRNA levels of MKK3/6 showed siRNA to MKK3/6 decreases its expression by 90% (P < .0002) compared to NT siRNA. A two-tailed t-test was used to test statistical significance. (d) AECs treated with CSE induced P-p38mapk, as seen previously, but treatment with siRNA to MKK3/6 and CSE did not reduce P38MAPK phosphorylation. (e) Quantitative densitometry (b and d) of P-p38MAPK over total p38MAPK shows a significant increase of activated p38MAPK when treated with CSE (P < .003); however, its phosphorylation is not inhibited when treated with siRNA to ASK1 or MKK3/6 and CSE.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Inhibition, Activation Assay, Expressing, Two Tailed Test

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: ASK1-Signolsome in AECs. (a) Immunofluorescence images of Cytokeritin-18 (CK-18) positive (green) AECs. Scale bar is set to 30 μM. (b) Western blot analysis—ASK1 (dimer 150 kDa top band, monomer 75 kDa lower band) and Trx are present in AECs, regardless of treatment. (c) Crystal violet stain (purple) was not expelled from AECs after CSE 1:10 treatment for 1 h. This was confirmed by plate reader analysis showing no difference in crystal violet intensity. (d) Cigarette smoke extract (CSE) treatment induced P-p38MAPK (P < .05), while NAC (P < .05) prevented its activation. (e) Densitometry of P-p38MAPK (1C) over total p38MAPK in CSE and CSE + NAC exposed AECs. A one-way ANOVA with the Tukey Multiple Comparisons Test was used to test statistical significance. (f) ASK1 pulldowns in columns 1, 2, 5, 6, 7, and 8 all show ASK1 present, but not Trx, in both control and CSE bead samples, while the Trx Pulldown in columns 3 and 4 show Trx present, but not ASK1, in both control and CSE bead samples.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Immunofluorescence, Western Blot, Staining, Activation Assay

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: Production and function of TGF-beta in AECs. (a) ELISA for TGF-beta conducted on AEC supernatant that had been stimulated with CSE for 48 h. AECs showed a significantly increased production of TGF-beta when stimulated with CSE (P < .05) and NAC can inhibit this production (P < .05). A one-way ANOVA with the Tukey Multiple Comparisons Test was used to test statistical significance. (b) AECs treated with 2 ng/mL of TGF-beta for up to 1 h caused activation of p38MAPK (two-fold) compared to control treated AECs. (c) AECs treated with CSE for 1 h significantly induced p38MAPK activation (P < 0.0001) while TGF receptor antagonist prevented p38MAPK activation (P = 0.03). (d) ELISA for TGF-beta conducted on amniotic fluid of TNIL or TL deliveries showed significant expression of TGF-beta at term (P < .05). A one-tailed t-test was used to test statistical significance. (e) Immunofluorescence colocalized TAK (red) and TAB1 (green) inside control treated AECs. White lines represent regions of interest to look for TAK1-TAB1 colocalization. Both cells showed overlapping line graphs of TAK1-TAB1 documenting colocalization within the cytoplasm. Scale bar is set to 30 μM. (f) Western blot analysis confirmed that TAB1 can be significantly (P = .047) activated by CSE and antioxidant NAC can significantly prevent this activation (P < = .057). (g) Quantitative densitometry of P-TAB1 over actin for panel f showing statistical significance. A one-way ANOVA with the Tukey Multiple Comparisons Test was used to test statistical significance.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Expressing, One-tailed Test, Immunofluorescence, Western Blot

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: Inhibition of TAK1 does not inhibit p38MAPK activation. (a) mRNA levels of TAK1 showed siRNA to TAK1 decrease its expression by 91% (P = .003) but NT siRNA does not decrease TAK1 gene expression 6% (P = .58). A two-tailed t-test was used to test statistical significance. (b) AECs treated with CSE and siRNA to TAK1 did not significantly reduce p38MAPK phosphorylation. (c) Quantitative densitometry (b) of P-p38MAPK over total p38MAPK showed significant increase of p38MAPK phosphorylation in AECs when treated with CSE (P = .001) and NT siRNA (P = .001); however, there was no significant difference of activated p38MAPK phosphorylation when treated with siRNA to TAK1 + CSE (P = 1.00). Surprisingly, NT siRNA + CSE also significantly upregulated p38MAPK activation compared to CSE alone (P = .006). A two-tailed t-test was used to test statistical significance.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Inhibition, Activation Assay, Expressing, Two Tailed Test

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: OS induces a TGF-beta–TAB1-dependent activation of p38MAPK. (a) mRNA levels of TAB1 showed siRNA to TAB1 decrease its expression by 91% (P < .0001) compared to NT siRNA. (b) AECs treated with CSE and siRNA to TAB1 significantly reduce p38MAPK phosphorylation (P = .0295). A two-tailed t-test was used to test statistical significance. (c) Quantitative densitometry (b) of P-p38MAPK over total p38MAPK shows a significant decrease of activated p38MAPK phosphorylation when treated with siRNA to TAB1 and CSE (P = .0295). A two-tailed t-test was used to test statistical significance.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Activation Assay, Expressing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Macrophage Cx43 Is Necessary for Fibroblast Cytosolic Calcium and Lung Fibrosis After Injury

doi: 10.3389/fimmu.2022.880887

Figure Lengend Snippet: P2rx4 is necessary for lung fibrosis. (A) qPCR for collagen genes and P2rx4 in primary mouse lung fibroblasts isolated from mice with fibroblast-specific P2rx4 deletion. N=3 mice. P values are for unpaired one-tailed t-tests. (B) qPCR for collagen genes in human (upper) and mouse (lower) primary lung fibroblasts treated with ATPγS. N=3 separate individuals and n=3 separate mice. P values are for unpaired two-tailed t-tests. (C) Left: Sirius red staining of lungs at 21 days post bleomycin injury in Col1a2-CreERT2 : P2rx4 fl/fl or Col1a2-CreERT2 controls. Representative images are shown. Quantification is for n=3 mice. Scale bar=50μm. P value is for unpaired two-tailed t-test. Right: Hydroxyproline assays of whole lung collagen from mice with fibroblast-specific P2rx4 deletion with two different Cre drivers as indicated, and controls. N=3-10 as shown. P values are for one-way ANOVA followed by Sidak’s multiple comparison test. qPCR is for P2rx4 in fibroblasts isolated from Pdgfrb-Cre: P2rx4 fl/fl or Pdgfrb-Cre controls. N=6 mice in each group and P value is for Mann-Whitney test. (D) Western Blot of phospho-p38 and total p38 for fibroblasts isolated from Pdgfrb-Cre : P2rx4 fl/fl or Pdgfrb-Cre controls. Quantification shows the ratio of P-p38 to total p38 by densitometry. P value is shown for unpaired one-tailed t-test and n=3 mice. (E) Model of fibroblast activation by paracrine ATP derived from monocyte-derived macrophages in lung fibrosis.

Article Snippet: Thereafter, the membranes were incubated with primary antibodies: Phospho-p38 MAPK (1:1000, Cell Signaling Technology, 9211) and p38 MAPK (1:1000, Cell Signaling Technology, 9212).

Techniques: Isolation, One-tailed Test, Two Tailed Test, Staining, Comparison, MANN-WHITNEY, Western Blot, Activation Assay, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: The critical role of the proto-oncogene c- Kit in TSC renal cystogenesis

doi: 10.1038/s44321-025-00360-x

Figure Lengend Snippet: ( A ) M-1 cells transfected with Foxi1 expression vector have significantly elevated levels of c-Kit transcript. The data represent the results of 3 independent transfection studies ( Foxi1 expression in Control vs Foxi1 transfected M-1 cells using a one-tailed t-test analysis p = 0.043411; * p < 0.04. c-Kit expression in Foxi1 vs Control transfected M-1 cells using a two-tailed t-test analysis p = 0.000326; ** p < 0.0003). Results are presented as average ± SD. ( B ) Overexpression of Foxi1 and c-Kit (Fig. ) lead to increased cell proliferation (the data represent the results of 2 independent experiments ( n = 3/cell line/timepoint/experiment) (Control vs c-Kit p = 0.00290213; Control vs Foxi1 p = 0.00436692; * p < 0.01). Results are presented as average ± SD. ( C ) Enhanced activation/phosphorylation of ERK1/2 and RSK1 kinases. ( D ) Cells lining the cystic epithelium co-express c-KIT (red staining indicated by white arrow) and PCNA (green staining indicated by yellow arrow). “C” represents cysts. Scale bar equals 20 μm. .

Article Snippet: ERK1/2 (rabbit polyclonal) , Cell Signaling Technology , 4695S.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, One-tailed Test, Two Tailed Test, Over Expression, Activation Assay, Phospho-proteomics, Staining

Journal: EMBO Molecular Medicine

Article Title: The critical role of the proto-oncogene c- Kit in TSC renal cystogenesis

doi: 10.1038/s44321-025-00360-x

Figure Lengend Snippet: RAS-MAPK signaling and TSC2 phosphorylation/inactivation were examined in the kidneys of WT and Tsc1 - KO mice. ( A ) Phosphorylation of ERK1/2, RSK1, and AKT were compared in WT and 28- and 45-day-old Tsc1 - KO mice ( n = 6/group). Image J analysis of the results (histogram) indicates that the phosphorylation of all three kinases is significantly elevated in day 28 kidneys of Tsc1-KO compared to WT mice (pERK1/2/ERK1/2, p = 0.009406302; pAKT/AKT, p = 0.007124116; and pRSK1/RSK1, p = 0.0003883); while in day 45 samples only the phosphorylation of ERK1/2 and RSK1 are significantly higher in the kidneys of Tsc1 - KO than that of WT mice (pERK1/2/ERK1/2, p = 0.016311363; and pRSK1/RSK1, p = 0.00644616). Results are presented as average ± SD. ( B ) Immunohistochemical labeling of p-ERK1/2, p-RSK1, and p-AKT in 45-day-old WT and Tsc1-KO mice. An increase in labeling can be seen within the cyst epithelium for all three phosphorylated kinases. “C” represents cysts. Scale bar equals 20 μm. ( C ) Phosphorylation of TSC2 by ERK1/2, AKT and RSK1 leads to its inactivation and allows unregulated mTORC1 driven cell proliferation (see the results in Fig. ). Phosphorylation of TSC2 on Serine residues 664, 939, and 1798 was compared in WT and Tsc1 - KO mice. Image J analysis of the results (histogram) indicates that the phosphorylation raito of 664Ser p-TSC2/TSC2 is significantly ( p = 1.78005 × 10 −5 ) elevated in the kidneys of Tsc1-KO ( n = 6) compared to WT ( n = 8) mice at 28 days. Image J analysis of TSC2 on day 45 indicates that all 3 sites are significantly more phosphorylated ( n = 8; 664Ser p-TSC2, p = 5.8533 × 10 −5 ; 939Ser p-TSC2, p = 0.032170819; and 1798Ser p-TSC2, p = 0.0197) in Tsc1-KO ( n = 8) compared to WT ( n = 8) mice. Results are presented as average ± SD. .

Article Snippet: ERK1/2 (rabbit polyclonal) , Cell Signaling Technology , 4695S.

Techniques: Phospho-proteomics, Immunohistochemical staining, Labeling

Journal: EMBO Molecular Medicine

Article Title: The critical role of the proto-oncogene c- Kit in TSC renal cystogenesis

doi: 10.1038/s44321-025-00360-x

Figure Lengend Snippet: ( A ) Levels of phosphorylated and total ERK1/2, RSK1 and AKT were compared in WT ( n = 6), Tsc1-KO ( n = 6), and Tsc1/c-Kit-dKO ( n = 6) mice. Image J analysis of phosphorylated to total activated kinases indicates that the Tsc1- KO mice have significantly elevated levels of AKT ( p = 0.010332); and RSK1 ( p = 0.00378008) compared to Tsc1/c-Kit - dKO mice; while comparing the phosphorylated to total activated kinase levels in Tsc1 - KO and WT mice denotes significant changes in AKT ( p = 0.024352) and RSK1 ( p = 0.006182228) content. Results are presented as average ± SD. ( B ) Immunohistochemical labeling of p-ERK1/2, p-RSK1, and p-AKT in WT, Tsc1-KO , and Tsc1/c-Kit-dKO mice. “C” represents a cyst. Scale bar equals 20 μm. ( C ) Comparison of the renal content of phosphorylated TSC2 in WT ( n = 6), Tsc1-KO ( n = 6), and Tsc1/c-Kit- dKO ( n = 6) mice was conducted by western blot followed by Image J analysis. Quantitative analysis of the results indicates that the Tsc1-KO mice have significantly elevated levels of 644Ser p-TSC2 ( p = 0.004555628), 939Ser p-TSC2 ( p = 0.001481909), and 1798Ser p-TSC2 ( p = 0.010024844) compared to Tsc1/c-Kit - dKO mice; while comparing the phosphorylated to total activated TSC1 levels in Tsc1 - KO and WT mice indicates significant changes in 644Ser p-TSC2 ( p = 0.012737439) and 939Ser p-TSC2 ( p = 0.010559358) content. All statistics of Image J results were conducted using T-test analysis. Results are presented as average ± SD. .

Article Snippet: ERK1/2 (rabbit polyclonal) , Cell Signaling Technology , 4695S.

Techniques: Immunohistochemical staining, Labeling, Comparison, Western Blot

Journal: bioRxiv

Article Title: Early life stress dysregulates kappa opioid receptor signaling within the lateral habenula

doi: 10.1101/2020.07.15.202614

Figure Lengend Snippet: A. Example images of non-MD (left) and MD (right) immunolabeling for LHb neurons (NeuN, red, top), Dyn-A(1-8) peptide (green, middle) and merged overlay images (bottom panel). Scale bar= 50μm. MD significantly increased average LHb Dyn-A density compared to non-MD controls (2-tailed unpaired t-test, t(17)=4.6, **p<0.001). B. LHb of MD rats had significantly lower abundance of opioid peptide receptor, kappa 1 (oprk1) mRNA expression compared to non-MD controls (unpaired student t-test, t(18)=3.4, p<0.01). C. inset: Example WB samples for ph-p38 (top) and total p38 (bottom) of LHb tissues across ACSF controls (-) and treatment with U50,488 (+) from non-MD/MD groups. Top graph: U50,488 significantly decreased ph-p38 in the LHb of MD rats (two-tailed unpaired t-test, t(6)=2.9, p<0.05) but only a trending effect was seen in the LHb of non-MD rats (one-tailed unpaired t-test, t(6)=1.6 p=0.081). ph-p38 levels in the LHb of MD+U50,488 group were significantly lower than non-MD+U50,488 group (one-tailed unpaired t-test, t(6)=2.2 p<0.05). All WB data: 3-4 rats each treatment. # p<0.1, * p<0.05, **p<0.01, *** p<0.0001. Middle graph: U50,488 significantly induced total p38 expression in the LHb of non-MD rats (one-tailed Mann Whitney test, p<0.05), while U50,488 incubation had no significant effect on the total p38 expression in the LHb of MD rats (two-tailed unpaired t-test, t(4)=0.9, p>0.05 n.s.). LHb of MD controls had significantly higher levels of total p38 expression compared to non-MD controls (two-tailed unpaired t-test, t(5)=3.4 p<0.05). Bottom graph: U50,488 significantly reduced ph-p38/p38 ratio in LHb of non-MD (two-tailed unpaired t-test, t(5)=3.4, p<0.05) and MD (two-tailed unpaired t-test, t(5)=3.5, p<0.05) rats. Additionally, LHb of MD rats had significantly lower Ph38/p38 compared non-MD controls (two-tailed, unpaired t-test t(6)=2.9, p<0.05). The ratio in LHb of MD+ACSF (MD controls) was trending higher than non-MD+U50,488 (2-tailed unpaired t-test, t(5)=2.1, p=0.086).

Article Snippet: Membranes were then rinsed and blocked with 5% BSA for 1hr prior to overnight incubation (4°C) with monoclonal rabbit anti-Phospho-p38 MAPK (Thr180/Tyr182) (D3F9)-XP (Cell signaling, 4511T, 1:1,000) or monoclonal rabbit anti-p38 MAPK (D13E1) XP (Cell signaling, 8690T, 1:1000).

Techniques: Immunolabeling, Expressing, Two Tailed Test, One-tailed Test, MANN-WHITNEY, Incubation

Journal: bioRxiv

Article Title: MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

doi: 10.1101/2023.01.23.524851

Figure Lengend Snippet: a,b) Quantification of nuclear pBrd4 levels by confocal IF with anti-pBrd4 (green) and DAPI staining of primary microglial cells pre-treated with 10 μM C13 (or DMSO) for 1 h and stimulated or not with 1 μg/mL LPS for 4 h. The data and images shown correspond to one experiment (N=20 analyzed images per condition) and are representative of two independent experiments. Scale bar: 10 μm. Student’s t-test (between two groups as indicated), unpaired, one-tailed. c,d) Quantification (c) and representative image (d) of Western blot analyses with anti-pBrd4 and anti-Brd4 (normalized to Brd4 or α-tubulin signal, respectively) in lysates of WT and MOK-KO SIM-A9 cells stimulated with 1 μg/mL LPS for 1 h. Data are from three independent experiments (N=3). Student’s t-test, unpaired, one-tailed. e,f) Quantification (e) and representative images (f) of confocal IF analyses of nuclear pBrd4 levels with anti-pBrd4 (green) and DAPI staining of WT and MOK-KO SIM-A9 cells stimulated or not with 1 μg/mL LPS for 1 h. Signal was enhanced by 50% in all four images in the first row for better visualization. Images are from one out of three independent experiments (N=3). Scale bar: 10 μm. Student’s t-test, unpaired, two-tailed. g) ChIP-PCR assessment of Brd4-binding to specific cytokine promoters. Represented data are relative values (percentage of input) of PCR products for IL-6, IFNβ and TNFα promoters after ChIP assay with either anti-Brd4 or IgG control antibodies with chromatin isolated from WT or MOK-KO cells treated or not with 1 μg/mL of LPS for 1 hour (N=4). Student’s t-test, paired, one-tailed. Data are the mean -/+ S.E.M. from ‘N’ independent experiments, and *P<0.05, **P<0.01 and ***P<0.001. NS: not significant.

Article Snippet: For intracellular labelling of bi-phosphorylated MOK protein (bpMOK), cells were fixed and permeabilized with the Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences, cat. 554722) according to the manufacturer instructions and stained with anti-MOK antibody bi-phosphorylated (pThr159+pTyr161, StressMarq, cat. SPC-1030), followed by donkey anti-rabbit IgG-Alexa Fluor 488 (Invitrogen, cat. A-21206).

Techniques: Staining, One-tailed Test, Western Blot, Two Tailed Test, Binding Assay, Control, Isolation

Journal: bioRxiv

Article Title: Early life stress dysregulates kappa opioid receptor signaling within the lateral habenula

doi: 10.1101/2020.07.15.202614

Figure Lengend Snippet: A. Example images of non-MD (left) and MD (right) immunolabeling for LHb neurons (NeuN, red, top), Dyn-A(1-8) peptide (green, middle) and merged overlay images (bottom panel). Scale bar= 50μm. MD significantly increased average LHb Dyn-A density compared to non-MD controls (2-tailed unpaired t-test, t(17)=4.6, **p<0.001). B. LHb of MD rats had significantly lower abundance of opioid peptide receptor, kappa 1 (oprk1) mRNA expression compared to non-MD controls (unpaired student t-test, t(18)=3.4, p<0.01). C. inset: Example WB samples for ph-p38 (top) and total p38 (bottom) of LHb tissues across ACSF controls (-) and treatment with U50,488 (+) from non-MD/MD groups. Top graph: U50,488 significantly decreased ph-p38 in the LHb of MD rats (two-tailed unpaired t-test, t(6)=2.9, p<0.05) but only a trending effect was seen in the LHb of non-MD rats (one-tailed unpaired t-test, t(6)=1.6 p=0.081). ph-p38 levels in the LHb of MD+U50,488 group were significantly lower than non-MD+U50,488 group (one-tailed unpaired t-test, t(6)=2.2 p<0.05). All WB data: 3-4 rats each treatment. # p<0.1, * p<0.05, **p<0.01, *** p<0.0001. Middle graph: U50,488 significantly induced total p38 expression in the LHb of non-MD rats (one-tailed Mann Whitney test, p<0.05), while U50,488 incubation had no significant effect on the total p38 expression in the LHb of MD rats (two-tailed unpaired t-test, t(4)=0.9, p>0.05 n.s.). LHb of MD controls had significantly higher levels of total p38 expression compared to non-MD controls (two-tailed unpaired t-test, t(5)=3.4 p<0.05). Bottom graph: U50,488 significantly reduced ph-p38/p38 ratio in LHb of non-MD (two-tailed unpaired t-test, t(5)=3.4, p<0.05) and MD (two-tailed unpaired t-test, t(5)=3.5, p<0.05) rats. Additionally, LHb of MD rats had significantly lower Ph38/p38 compared non-MD controls (two-tailed, unpaired t-test t(6)=2.9, p<0.05). The ratio in LHb of MD+ACSF (MD controls) was trending higher than non-MD+U50,488 (2-tailed unpaired t-test, t(5)=2.1, p=0.086).

Article Snippet: Membranes were then rinsed and blocked with 5% BSA for 1hr prior to overnight incubation (4°C) with monoclonal rabbit anti-Phospho-p38 MAPK (Thr180/Tyr182) (D3F9)-XP (Cell signaling, 4511T, 1:1,000) or monoclonal rabbit anti-p38 MAPK (D13E1) XP (Cell signaling, 8690T, 1:1000).

Techniques: Immunolabeling, Expressing, Two Tailed Test, One-tailed Test, MANN-WHITNEY, Incubation

Journal: bioRxiv

Article Title: JG2: an updated version of the Japanese population-specific reference genome

doi: 10.1101/2024.11.01.621223

Figure Lengend Snippet: Three Japanese male volunteers (jg1a, jg1b, jg1c) provided the data for JG2. High-coverage sequencing and optical genome mapping data were generated: PacBio CLRs, Hi-C reads, Bionano optical genome maps, ONT nanopore long reads, Illumina mate-pair reads, and Illumina paired-end reads. (A) Phased genome assemblies and scaffolding were performed for each individual using PacBio CLRs and Hi-C reads. Meta-assemblies were created within each individual. (B) Bionano optical genome maps were assembled, and PacBio/Hi-C-based assemblies were scaffolded with the optical genome maps in each individual. In addition, ONT long reads were assembled within each individual, then meta-assembled with the PacBio/Hi-C/Bionano-based assemblies, resulting in a draft assembly for each individual. (C) Draft assemblies for the three individuals were meta-assembled. White boxes indicate raw data, gray boxes indicate intermediate assemblies, and black rounded boxes indicate software or operations. CLR, continuous long reads; ONT, Oxford Nanopore Technology.

Article Snippet: For each individual, we obtained over 120× PacBio CLR, two sets of over 49× Hi-C reads (using MboI and HindIII enzymes), one or two sets of over 120× Bionano optical genome maps (using DSL-1 or BspQI and BssSI enzymes), one set of ONT long reads, two sets of mate-pair read, and two sets of paired-end reads (Illumina HiSeq 162-bp and 259-bp reads).

Techniques: Sequencing, Generated, Hi-C, Scaffolding, Software