Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: a Representative Masson’s Trichrome staining in tracheal sections at day 14. Scale bars: 200 μm. Cartilage rings are labelled C1–C6. b – e Quantification of collagen deposition over WT PBS d14 (b) or over WT ALT d14 (d), and gland numbers from C1-C7 cartilages ( c , e ). Group sizes: n = 7, 7, 5, 4, 7 ( b , c ) and n = 4, 5, 3 ( d , e ) mice, from left to right. f Experimental schema. g Representative images of lineage-labelled Sox9-tdT + cells among BCAM + BCs on the SAE at day 14. Scale bar: 200 μm. Areas outlined (a, b) are shown below (Scale bars: 50 μm). Cartilage rings are labelled C1–C7. CC Cricoid cartilage. h , i Quantification of Sox9-tdT + BCAM + cells in the SAE per high power field (HPF) ( h ) and as a percentage of total BCAM + cells ( i ) in ALT-injured trachea at day 14. Group sizes: n = 7, 15, 6 mice, from left to right. j , k Quantification of Sox9-tdT + BCAM + cells in the SAE per HPF ( j ) and as a percentage of total BCAM + cells ( k ) in influenza-injured trachea at 7 days post infection. Group sizes: n = 3, 6, 8 mice, from left to right. l , m Representative images ( l ) and quantification ( m ) of BCAM and SOX9 staining in tracheal sections at day 14. Scale bars: 20 μm. Group sizes: n = 5, 6, 4 mice, from left to right. Two-tailed unpaired Mann-Whitney U test ( b – e , m ). Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction ( h – k ). The dashed outline ( g , i ) marks the border between SAE and SM. Box plots ( b – e , h – k , m ) show median (center line), 25th–75th percentiles (box), and min–max values (whiskers). Each data point represents the average of 3–4 images from one mouse, except for the glandular number ( c , e ), which represents the total glands per trachea measured by tiled imaging. See “Methods”. Panel f was created in BioRender. Lee, M. ( https://BioRender.com/dg3i7rm ). Source data are provided as a Source Data file.

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques: Staining, Infection, Two Tailed Test, MANN-WHITNEY, Imaging

Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: a Representative images of DCLK1 and BCAM staining in tracheal sections from the indicated Sox9-tdT reporter strains at day 14. Sox9-tdT + BCAM + cells (white arrow). Scale bars: 50 µm. b Quantification of Sox9-tdT + BCAM + cells in the SAE from the indicated strains at day 14. Group sizes: n = 5, 6, 12, 5 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. c , d Combined in situ hybridization (ISH) for Oxgr1, Krt5 and Krt8 , and immunofluorescence for SOX9 and α-SMA in tracheal sections from WT mice with or without ALT treatment ( Krt5 for BCs, Krt8 for differentiated cells, SOX9 for SMG progenitor cells, and α-SMA for SMG myoepithelial cells). Oxgr1 + SOX9 + cells (green arrows). Scale bars: 20 μm. SMG Submucosal gland. ( e ) Quantification of Oxgr1 + cells in the specified EpC subsets. Group sizes: n = 7, 16, 8, 13, 6, 10, 7, 8 mice, from left to right. Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction. f Quantification of Oxgr1 + cells among the Krt5 + or Krt8 + cells. Group sizes: n = 12, 11, 12, 7 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. g Experimental schema. h , i Representative images ( h ) and quantification ( i ) of BCAM and SOX9 staining in tracheal sections from the indicated strains at day 14. Scale bars: 20 μm. Group sizes: n = 5, 8, 7, 5 mice, from left to right. Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction. The dashed line marks the border between SAE and SM. Box plots ( b , e , f , i ) show median (center line), 25th–75th percentiles (box), and min–max values (whiskers). Each data point represents the average of 3–4 images from one mouse. See “Methods”. Panel g was created in BioRender. Lee, M. ( https://BioRender.com/dg3i7rm ). Source data are provided as a Source Data file.

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, In Situ Hybridization, Immunofluorescence

Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: a Experimental schema. b Principal-component analysis (PCA) of the top 500 most variable genes expressed in the two groups. Numbers in parentheses indicate the percent variance captured by each PC. c Relative expression of Sox9 and canonical BC genes. d Heatmap of 1443 differentially expressed genes (DEGs) following unsupervised hierarchical clustering, defined by |log2FoldChange | > 0.58 and adjusted p-values < 0.05 with multiple testing correction using the Benjamini–Hochberg method ( e ) GSEA enrichment plots for two of the most overrepresented gene sets. Gene set enrichment analysis (GSEA) was performed using a weighted Kolmogorov–Smirnov–like statistic to calculate enrichment scores. Statistical significance was assessed by permutation testing, and nominal P values were derived from the permutation-based null distribution. Normalized enrichment scores (NES) were calculated to account for differences in gene set size, and multiple hypothesis testing was controlled using the Benjamini–Hochberg false discovery rate (FDR) correction. f – h Heatmaps of DEGs related to alarmins and inflammatory genes ( f ), collagen component genes ( g ), and ciliogenesis and cilia assembly genes ( h ). i , j Representative images ( i ) and quantification ( j ) of acetylated tubulin (Ac-tub) staining on tracheal sections from the indicated strains at day 14. Scale bars: 20 μm. The dashed line marks the border between SAE and SM. Group sizes: n = 7, 8 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. Box plots ( j ) show median (center line), 25th–75th percentiles (box), and min–max values (whiskers). Each data point represents the average of 3–4 images from one mouse. See Methods. Panel a was created in BioRender. Lee, M. ( https://BioRender.com/dg3i7rm ). Source data are provided as a Source Data file.

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques: Expressing, Derivative Assay, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: a Combined ISH for Pou2f3 , and immunofluorescence for E-Cadherin in human sinonasal tissue from healthy control (HC) and CRSwNP. Pou2f3 + cells (yellow arrow). Scale bars: 20 µm. b , c Quantification of Pou2f3 + tuft cells ( b ) and E-Cadherin staining ( c ) in the SAE across human sinonasal tissues. Group sizes: n = 6, 11, 27 independent patients, from left to right. Two-tailed unpaired Mann-Whitney U test ( b ). Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction ( c ). d Spearman’s correlation analysis (two-sided) between E-Cadherin levels and Pou2f3 + tuft cell numbers in patients with CRSwNP ( n = 27, independent patients). No multiple comparison adjustment was applied. e Representative images of E-Cadherin, α-SMA, and SOX9 staining in human sinonasal mucosa from CRS patients and healthy control (HC). SOX9 + α-SMA + cells (yellow arrow). Scale bars: 20 µm. f Quantification of SOX9 + and/or α-SMA + cells in the SAE from the indicated groups. Group sizes: n = 6, 11, 27 independent patients, from left to right. Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction. g , h Spearman’s correlation analysis (two-sided) between SOX9 + cell numbers and E-Cadherin intensity ( g ) and α-SMA + cell numbers ( h ) in patients with CRSwNP ( n = 27 independent patients). No multiple comparison adjustment was applied. i Spearman’s correlation analysis (two-sided) between E-Cadherin levels and SOX9 + α-SMA + cell numbers in patients with CRSwNP ( n = 27 independent patients). No multiple comparison adjustment was applied. The Spearman r values and the corresponding P values were calculated ( g – i ). j SOX9 progenitor cell signature module score in basal EpCs from the Wang scRNA-seq dataset (HRA000772) across the patients. One-sided Wilcoxon rank sum test was used. No multiple comparison adjustment was applied. The dashed line marks the border between SAE and SM. Box plots ( b , c , f ) show median (center line), 25th–75th percentiles (box), and min–max values (whiskers). Each data point represents the average of 3–4 images from one patient. See “Methods”. Source data are provided as a Source Data file.

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques: Immunofluorescence, Control, Staining, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: (Left) In homeostasis, basal cells are the dominant progenitor of all ciliated and secretory epithelial cells on the SAE. (Right) In the setting of tissue damage elicited by allergens or by influenza ( 1 ), tuft cells generate CysLTs (2). Leukotriene E 4 (LTE 4 ) activates OXGR1 (also known as CysLTR3 and GPR99) on SOX9 + SMG progenitors ( 3 ). This results in airway remodeling including SMG hyperplasia ( 4 ) and submucosal collagen deposition ( 5 ). SOX9 + progenitors regenerate the SAE but allow persistent barrier leak ( 6 ), bringing a program that is rich in mesenchymal genes and deficient in ciliogenesis genes. This leads to an SAE with reduced ciliated cells, reduced E-Cadherin, and increased α-SMA ( 7 ). This figure was created in BioRender. Barrett, N. ( https://BioRender.com/d9cr3ur ).

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques:

Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: a Experimental schema. b Representative images of H&E staining in tracheal sections from the indicated strains at day 1. c , d Quantification of DAPI + cells ( c ) and epithelial height ( d ) from the WT and Pou2f3 -/- at the indicated time points. Group sizes: n = 3, 4, 12, 11, 6, 7 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. e Representative images of TUNEL or p-γ-H2A.X staining in tracheal sections from the indicated strains at day 14. f Quantification of TUNEL + (top panel) or p-γ-H2A.X + (bottom panel) cells from the WT and Pou2f3 -/- at the indicated time points. Group sizes: n = 3, 4, 12, 11, 6, 7 mice, from left to right. Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction. g , h Representative images ( g ) and quantification ( h ) of FITC-dextran in tracheal sections from the WT and Pou2f3 -/- at day 11. Group sizes: n = 6, 7, 6 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. i , j Representative images ( i ) and quantification ( j ) of CLDN1 staining in tracheal sections from the WT and Pou2f3 -/- at day 14. Group sizes: n = 6, 7, 6 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. k Experimental schema. l – n Representative images ( l ), and quantification of TUNEL + ( m ) and p-γ-H2A.X + ( n ) from the indicated strains. Group sizes: n = 4, 7 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. Scale bars: 50 μm ( b , e , g , i , l ). The dashed line marks the border between surface airway epithelium (SAE) and submucosa (SM). Box plots ( c , d , f , h , j , m , n ) show median (center line), 25th–75th percentiles (box), and min–max values (whiskers). Each data point represents the average of 3–4 images from one mouse, except FITC intensity ( h ), which represents total intensity per trachea measured by tiled imaging. See “Methods”. Panels a and k were created in BioRender. Lee, M. ( https://BioRender.com/dg3i7rm ). Source data are provided as a Source Data file.

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, TUNEL Assay, Imaging

Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: a Combined ISH for Pou2f3 , and immunofluorescence for E-Cadherin in human sinonasal tissue from healthy control (HC) and CRSwNP. Pou2f3 + cells (yellow arrow). Scale bars: 20 µm. b , c Quantification of Pou2f3 + tuft cells ( b ) and E-Cadherin staining ( c ) in the SAE across human sinonasal tissues. Group sizes: n = 6, 11, 27 independent patients, from left to right. Two-tailed unpaired Mann-Whitney U test ( b ). Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction ( c ). d Spearman’s correlation analysis (two-sided) between E-Cadherin levels and Pou2f3 + tuft cell numbers in patients with CRSwNP ( n = 27, independent patients). No multiple comparison adjustment was applied. e Representative images of E-Cadherin, α-SMA, and SOX9 staining in human sinonasal mucosa from CRS patients and healthy control (HC). SOX9 + α-SMA + cells (yellow arrow). Scale bars: 20 µm. f Quantification of SOX9 + and/or α-SMA + cells in the SAE from the indicated groups. Group sizes: n = 6, 11, 27 independent patients, from left to right. Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction. g , h Spearman’s correlation analysis (two-sided) between SOX9 + cell numbers and E-Cadherin intensity ( g ) and α-SMA + cell numbers ( h ) in patients with CRSwNP ( n = 27 independent patients). No multiple comparison adjustment was applied. i Spearman’s correlation analysis (two-sided) between E-Cadherin levels and SOX9 + α-SMA + cell numbers in patients with CRSwNP ( n = 27 independent patients). No multiple comparison adjustment was applied. The Spearman r values and the corresponding P values were calculated ( g – i ). j SOX9 progenitor cell signature module score in basal EpCs from the Wang scRNA-seq dataset (HRA000772) across the patients. One-sided Wilcoxon rank sum test was used. No multiple comparison adjustment was applied. The dashed line marks the border between SAE and SM. Box plots ( b , c , f ) show median (center line), 25th–75th percentiles (box), and min–max values (whiskers). Each data point represents the average of 3–4 images from one patient. See “Methods”. Source data are provided as a Source Data file.

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques: Immunofluorescence, Control, Staining, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: Tuft cells shape airway remodeling by eliciting OXGR1- and SOX9-dependent stem cell programs

doi: 10.1038/s41467-026-70763-y

Figure Lengend Snippet: a Representative images of DCLK1 and BCAM staining in tracheal sections from the indicated Sox9-tdT reporter strains at day 14. Sox9-tdT + BCAM + cells (white arrow). Scale bars: 50 µm. b Quantification of Sox9-tdT + BCAM + cells in the SAE from the indicated strains at day 14. Group sizes: n = 5, 6, 12, 5 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. c , d Combined in situ hybridization (ISH) for Oxgr1, Krt5 and Krt8 , and immunofluorescence for SOX9 and α-SMA in tracheal sections from WT mice with or without ALT treatment ( Krt5 for BCs, Krt8 for differentiated cells, SOX9 for SMG progenitor cells, and α-SMA for SMG myoepithelial cells). Oxgr1 + SOX9 + cells (green arrows). Scale bars: 20 μm. SMG Submucosal gland. ( e ) Quantification of Oxgr1 + cells in the specified EpC subsets. Group sizes: n = 7, 16, 8, 13, 6, 10, 7, 8 mice, from left to right. Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction. f Quantification of Oxgr1 + cells among the Krt5 + or Krt8 + cells. Group sizes: n = 12, 11, 12, 7 mice, from left to right. Two-tailed unpaired Mann-Whitney U test. g Experimental schema. h , i Representative images ( h ) and quantification ( i ) of BCAM and SOX9 staining in tracheal sections from the indicated strains at day 14. Scale bars: 20 μm. Group sizes: n = 5, 8, 7, 5 mice, from left to right. Kruskal–Wallis test followed by Dunn’s multiple-comparisons test with Holm–Bonferroni correction. The dashed line marks the border between SAE and SM. Box plots ( b , e , f , i ) show median (center line), 25th–75th percentiles (box), and min–max values (whiskers). Each data point represents the average of 3–4 images from one mouse. See “Methods”. Panel g was created in BioRender. Lee, M. ( https://BioRender.com/dg3i7rm ). Source data are provided as a Source Data file.

Article Snippet: Female (12 weeks- to 14 weeks) and male (10 weeks- to 12 weeks) mice of the following genotypes and strains were used: C57BL/6 (Charles River Laboratories, CRL# 027), Oxgr1 -/- (KOMP, stock number 048933-UCD), Pou2f3 -/- (The Jackson Laboratory, stock number 037040), Cysltr1 -/- (kindly provided by Dr. Lora Bankova, BWH, Boston), Cysltr2 -/- (kindly provided by Dr. Joshua Boyce, BWH, Boston), Krt5 CreERT2 (The Jackson Laboratory, stock number 029155), Pou2f3 CreERT2 (The Jackson Laboratory, stock number 037511), Sox9 CreERT2 (The Jackson Laboratory, stock number 035092), Sox9 fl/fl (The Jackson Laboratory, stock number 013106), Oxgr1 fl/fl (Barrett Lab), Ltc4s fl/fl (kindly provided by Dr. Lora Bankova, BWH, Boston), and Ai9 (RCL-tdT) (The Jackson Laboratory, stock number 007909).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, In Situ Hybridization, Immunofluorescence

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) Timeline for conditional deletion of endothelial Ripk3 ( Ripk3 iECKO ) in 8-week-old mice and subsequent baseline or intestinal I/R injury-induced vascular permeability measurements using Evans blue dye (EBD) in 12-week-old mice. ( B ) Baseline vascular permeability measurements using EBD in Ripk3 WT and Ripk3 iECKO mice in the small intestine (n=5/group). ( C ) Left: schematic displaying small intestinal regions where vascular permeability was measured after I/R injury. Right: small intestinal vascular permeability (fold change from each genotype’s own baseline) after 24 hr intestinal I/R injury in the injury region (I/R) and adjacent control regions (control, distal control) (n=6/group). ( D ) Representative images of FITC-dextran (70kDa) permeability (white arrows) in control regions of small intestinal tissue after 24 hr I/R injury. Scale bar: 100 μm. Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test ( B ) and two-way ANOVA ( C ). SMA: superior mesenteric artery.

Article Snippet: Ripk3 -floxed mice 5 , Cdh5(PAC)-Cre ERT2 mice (gift of Ralf Adams, Max Planck Institute for Molecular Biomedicine; Taconic; #13073) 27 , RIPK3-GFP mice ( Ripk3-gfp fl/fl ; The Jackson Laboratory; #030284) 28 , and ROSA mT/mG mice ( Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Lou /J ; The Jackson Laboratory; #007576;) 29 were previously described.

Techniques: Permeability, Control, Two Tailed Test

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) Schematic of mouse tissue collection and quantitative analysis of cytokines at 0 hr and 4 hr after I/R injury in Ripk3 WT and Ripk3 iECKO mice. ( B ) Baseline IL-6 levels (left) and fold change (from baseline) following 4 hr I/R injury (right) in small intestinal tissue of Ripk3 WT and Ripk3 iECKO mice (n=3–4/group). ( C ) Baseline IL-6 levels (left) and fold change (from baseline) following 4 hr I/R injury (right) in serum of Ripk3 WT and Ripk3 iECKO mice (n=3–4/group). ( D ) Schematic of IL-6 measurement in stimulated whole blood and MS1 endothelial cells (ECs). ( E and F ) Plasma IL-6 levels at 2 hr ( E ) and 4 hr ( F ) after ex-vivo stimulation of whole blood collected from Ripk3 WT and Ripk3 iECKO mice. At each time point, blood was stimulated with phosphate-buffered saline (PBS) (left) or lipopolysaccharide (LPS) (right); LPS data are graphed as fold change from PBS control treatments (n=3/group). ( G ) IL-6 levels in cell supernatants following stimulation of WT and Ripk3 -KO MS1 ECs with 20ng/mL TNFα for 0 hr and 24 hr (n=3/group). ( H ) Il6 transcripts measured by qRT-PCR in WT and Ripk3 -KO MS1 ECs following stimulation with 20ng/mL TNFα for 0 hr, 4 hr and 8 hr (n=4/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test ( B , C , E and F ) and two-way ANOVA ( G and H ).

Article Snippet: Ripk3 -floxed mice 5 , Cdh5(PAC)-Cre ERT2 mice (gift of Ralf Adams, Max Planck Institute for Molecular Biomedicine; Taconic; #13073) 27 , RIPK3-GFP mice ( Ripk3-gfp fl/fl ; The Jackson Laboratory; #030284) 28 , and ROSA mT/mG mice ( Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Lou /J ; The Jackson Laboratory; #007576;) 29 were previously described.

Techniques: Clinical Proteomics, Ex Vivo, Saline, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) Representative immunofluorescence images in grey scale depicting cytosolic versus nuclear NRF2 expression at baseline and 1 hr following 20ng/ml TNFα or tert-Butylhydroquinone (tBHQ) stimulation in WT and Ripk3 -KO MS1 ECs. Scale bar: 20 μm. ( B-D ) Mean NRF2 fluorescence intensity in cytosolic and nuclear compartments (left) and calculated NRF2 nuclear to cytosolic ratio (right) in WT and Ripk3 -KO MS1 ECs at baseline ( B ) or after 1 hr stimulation with TNFα ( C ) or tBHQ ( D ) (n=3/group). ( E-F ) Transcript expression following stimulation with 20ng/ml TNFα for 0 hr and 4 hr in WT and Ripk3 -KO MS1 ECs of the following genes: Hmox1 (heme oxygenase-1) ( E ) and Nqo1 [NAD(P)H quinone dehydrogenase 1] ( F ) (n=3–4/group). ( G ) Il6 transcript expression in Ripk3 -KO and Ripk3 -KO + NRF2 overexpression (OE) MS1 ECs following stimulation with 20ng/mL TNFα for 0 hr and 4 hr (n=3/group). ( H ) IL-6 levels in cell supernatants following stimulation of Ripk3 -KO and Ripk3 -KO+ NRF2 OE MS1 ECs with 20ng/mL TNFα for 0 hr, 4 hr and 24 hr (n=3/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test or two-way ANOVA ( B , C and D ) and two-way ANOVA ( E , F , G and H ).

Article Snippet: Ripk3 -floxed mice 5 , Cdh5(PAC)-Cre ERT2 mice (gift of Ralf Adams, Max Planck Institute for Molecular Biomedicine; Taconic; #13073) 27 , RIPK3-GFP mice ( Ripk3-gfp fl/fl ; The Jackson Laboratory; #030284) 28 , and ROSA mT/mG mice ( Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Lou /J ; The Jackson Laboratory; #007576;) 29 were previously described.

Techniques: Over Expression, Immunofluorescence, Expressing, Fluorescence, Two Tailed Test

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) VCAM-1 expression in the small intestine assessed by immunoblot in baseline Ripk3 WT and Ripk3 iECKO mice (n=7/group). ( B ) VCAM-1 expression assessed by immunoblot in WT and Ripk3 -KO MS1 endothelial cells (ECs) stimulated with 20ng/ml TNFα for 24 hr (n=4/group). ( C ) WT and Ripk3 -KO MS1 cells were stimulated with either PBS or 20ng/ml TNFα for 24 hr and immunostained for VCAM-1. White arrow heads in the insets indicate junctional localization of VCAM-1, Scale bar: 20 μm, inset = 10 μm. ( D ) VCAM-1 expression assessed by immunoblot in WT and Ripk3 -KO MS1 ECs stimulated with 20ng/ml TNFα for 24 hr in the presence of control IgG antibody or neutralizing IL-6 antibody (n=4/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test ( A ) and two-way ANOVA ( B and D ).

Article Snippet: Ripk3 -floxed mice 5 , Cdh5(PAC)-Cre ERT2 mice (gift of Ralf Adams, Max Planck Institute for Molecular Biomedicine; Taconic; #13073) 27 , RIPK3-GFP mice ( Ripk3-gfp fl/fl ; The Jackson Laboratory; #030284) 28 , and ROSA mT/mG mice ( Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Lou /J ; The Jackson Laboratory; #007576;) 29 were previously described.

Techniques: In Vivo, In Vitro, Expressing, Western Blot, Control, Two Tailed Test

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: RIPK3 protects against endothelial activation and vascular permeability in a mouse model of ischemia-reperfusion injury

doi: 10.1161/ATVBAHA.125.322977

Figure Lengend Snippet: ( A ) CD45 + leukocyte accumulation was assessed by immunostaining of small intestinal tissues from Ripk3 WT and Ripk3 iECKO mice following 24 hr I/R injury. Scale bar: 100 μm. ( B ) CD45 + cells, as in A, were quantified from multiple confocal Z-stack images taken from each mouse tissue by normalizing CD45 + expression area to total cell nuclear area in small intestines. Individual data points are averages of 2–4 imaged and quantified sections per mouse (n=5/group). ( C ) Representative images of immortalized BMA3.1A7 macrophages labeled with calcein-AM fluorescent dye (green) and incubated on top of a monolayer of WT and Ripk3 -KO MS1 endothelial cells (ECs) that were previously treated with 20ng/mL TNFα for 24 hr. Cells were washed multiple times before imaging. Scale bar: 20 μm. (D) The number of macrophages adherent to ECs was quantified in each acquired image (n=4 images/group). ( E ) Following imaging (as in C), cells were lysed with Triton-X, and fluorescence was measured using a plate reader. Values were normalized to readings from wells in which BMA3.1A7 cells were incubated with untreated WT MS1 ECs (dotted line) (n=3/group). ( F ) Timeline for detecting vascular permeability with Evans blue dye (EBD) after clodronate-liposome treatment and intestinal I/R injury; tamoxifen was administered at 8 weeks of age. ( G ) Small intestinal vascular permeability (fold change from each genotype’s own baseline) after 24 hr intestinal I/R injury; measurements are shown in the injury region (I/R) and control regions (control, distal control) following clodronate-liposome treatment (n=4–5/group). ( H ) CD45 + leukocyte accumulation was assessed by immunostaining in small intestinal tissues from Ripk3 WT and Ripk3 iECKO mice following 24 hr I/R injury and clodronate-liposome treatment. ( I ) CD45 + cells, as in G, were quantified from multiple confocal Z-stack images taken from each mouse; CD45 + expression area was normalized to total cell nuclear area in small intestines. Individual data points are averages of 3–4 imaged and quantified sections per mouse (n=3/group). Data represent mean ± SEM. P values were determined by two-tailed Student’s t -test ( B , D , E and I ) and two-way ANOVA ( G ).

Article Snippet: Ripk3 -floxed mice 5 , Cdh5(PAC)-Cre ERT2 mice (gift of Ralf Adams, Max Planck Institute for Molecular Biomedicine; Taconic; #13073) 27 , RIPK3-GFP mice ( Ripk3-gfp fl/fl ; The Jackson Laboratory; #030284) 28 , and ROSA mT/mG mice ( Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Lou /J ; The Jackson Laboratory; #007576;) 29 were previously described.

Techniques: Activation Assay, Permeability, Immunostaining, Expressing, Labeling, Incubation, Imaging, Fluorescence, Control, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

doi: 10.1074/jbc.m112.422295

Figure Lengend Snippet: FIGURE 1. Effect of Pak1 and Pak2 on mast cell maturation and antigen-mediated secretion. A, WT MC-9 mouse mast cell line lysates were subjected to immunoblotting and probed with either anti-Pak1 or anti-Pak2 antibodies. Membranes were exposed together on the same film for the same amount of time for semiquantitative analysis. 10-s and 1-min exposures are shown, with loading control actin. B, BMMC lysates from Pak1 knock-out mice and Pak2fl/fl infected with MSCV-Cre-ERT2 and treated with 250 nM 4-HT with actin loading control. C, loss of Pak1 or Pak2 does not affect expression of mast cell maturation markers. Mast cells were cultured for 4 weeks with cytokines, and expression of c-Kit and FcRI was measured by FACS. Double-positive cells (upper right quadrant) are mature mast cells. Data shown are representative of six independent lines from each genotype. D, -hexosaminidase release was measured in IgE-primed Pak1 andPak2knock-outBMMCsstimulatedwith10or30ng/mlDNP-HSAfor45min.Averagepercentagesofcontrolofatleastthreeindependentexperimentsrun in triplicate are shown here with standard deviations. FcRI-independent degranulation is shown with ionomycin treatment. In all conditions, the extent of degranulation is represented as the percentage of total -hexosaminidase activity in cells. *, p value 0.05, Wilcoxon signed-rank test. E, ELISA was performed for TNF- and IL-6 secreted by Pak1 knock-out and Pak2 knock-out BMMCs in response to 30 ng/ml DNP-HSA. Results are shown as average -fold change over WT cells for at least three independent experiments run in triplicate. *, p value 0.05, Wilcoxon signed-rank test.

Article Snippet: Cre Activation—A retroviral vector for Cre recombinase (MSCV-CRE-ERT2) under tamoxifen control was used to excise Pak2 (Addgene plasmid 22776) (15).

Techniques: Western Blot, Control, Knock-Out, Infection, Expressing, Cell Culture, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

doi: 10.1074/jbc.m112.422295

Figure Lengend Snippet: FIGURE 2. Effect of Pak1 and Pak2 on calcium signaling and PLC1. A, representative immunoblots for activated PLC1 in Pak1 and Pak2 knock-out BMMCs. IgE-primedWTandknock-outBMMCswerestimulatedwithDNP-HSA(100ng/ml)for3min,andlysatesweresubjectedtoimmunoblottingwithanti-phospho- PLC1 phospho-Tyr-783 (pPLCy1 pY783, middle panel) or anti-total PLC1 (bottom panel). Experiments were done on three different mouse BMMCs. Pak1 and Pak2 status is shown for each blot (top panel). B, IgE-primed WT, Pak1 knock-out and Pak2 knock-out BMMCs were loaded with Ca2-sensitive dye (Indo-1-AM) and suspended in Ca2-containing medium. After base-line collection on JSAN flow cytometer, FcRI was activated by the addition of 30 ng/ml DNP-HSA. The second stimulation peak is from thapsigargin treatment. Representative experiments of WT (black) versus Pak1 knock-out (gray) (left panel) and Pak2fl/fl (black) versusPak2knock-out(gray)(rightpanel)BMMCsareshown.C,mean(S.D.)stimulation(peakofratiominusbase-lineasthepercentageofWT)ofatleastthree independent experiments for Pak1 knock-out and Pak2 knock-out BMMCs. Pak1 knock-out, *, p value 0.05, Wilcoxon signed-rank test. Pak2 is not statistically different from WT. ns, not significant.

Article Snippet: Cre Activation—A retroviral vector for Cre recombinase (MSCV-CRE-ERT2) under tamoxifen control was used to excise Pak2 (Addgene plasmid 22776) (15).

Techniques: Western Blot, Knock-Out, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

doi: 10.1074/jbc.m112.422295

Figure Lengend Snippet: FIGURE 3. Effect of Pak1 and Pak2 on adhesion. A, the percentage of IgE-mediated adhesion in Pak1 knock-out BMMCs on fibronectin-coated plates with 1 and 10 ng/ml DNP-HSA (*, p value 0.05, Student’s t test). B, the percentage of IgE-mediated adhesion in Pak2 knock-out BMMCs (*, p value 0.05, Student’s t test). Data are shown as the mean S.D. of at least three independent experiments run in triplicate. Data were calculated as the percentage of adhesion relative to total cells in each well using calcein-AM viability dye.

Article Snippet: Cre Activation—A retroviral vector for Cre recombinase (MSCV-CRE-ERT2) under tamoxifen control was used to excise Pak2 (Addgene plasmid 22776) (15).

Techniques: Knock-Out

Journal: Journal of Biological Chemistry

Article Title: Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

doi: 10.1074/jbc.m112.422295

Figure Lengend Snippet: FIGURE 4. Effect of Pak2 on Rho GTPase activity. A, IgE-primed Pak2fl/fl and Pak2/ BMMCs were stimulated with 100 ng/ml DNP-HSA for 10 min, and lysates were cleared and incubated with Rhotekin RBD-GST beads (Millipore). RhoA-GTP was detected by anti-RhoA antibody, and 10% of input demonstrates equal loading. One representative Western blot of three experiments is shown, and densitometry of the -fold induction relative to unstimulated cells (all normalized to input) of three independent experiments is shown (*, p value 0.05, Student’s t test). B, activation status of MLC2, a downstream RhoA effector, at p-Thr-18/Ser-19inlysatesfrom10minofDNP-HSAstimulation.Theblotisrepresentativeofthreeseparateexperiments,anddensitometryshowsaverage-fold ( S.D.) change over stimulated wild-type cells normalized to total MLC2 for three independent experiments (*, p value 0.05, Student’s t test). pMLC2, phospho-MLC2. C, C3 exoenzyme, a RhoA inhibitor, was treated on Pak2fl/fl and Pak2/ BMMCs for 6 h without serum. A degranulation assay was performed with 30 ng/ml DNP-HSA. The graph is an average of three independent experiments run in triplicate with S.D. (*, p value 0.05, Pak2/ versus Pak2/ C3, Student’s t test). D, Rac1 and Cdc42 activity (GTP-bound isoforms) of Pak2fl/fl and Pak2/ BMMCs after DNP-HSA stimulation for 10 min (representative blot of three independent BMMCs). Total Rac1 and Cdc42 proteins for each sample before performing pulldown are detected with monoclonal anti-Rac1 and polyclonal anti-Cdc42.

Article Snippet: Cre Activation—A retroviral vector for Cre recombinase (MSCV-CRE-ERT2) under tamoxifen control was used to excise Pak2 (Addgene plasmid 22776) (15).

Techniques: Activity Assay, Incubation, Western Blot, Activation Assay, Degranulation Assay

Journal: Journal of Biological Chemistry

Article Title: Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

doi: 10.1074/jbc.m112.422295

Figure Lengend Snippet: FIGURE 5. Effect of Pak2 on MAPK activity. A, IgE-primed Pak2fl/fl and Pak2/ BMMCs were stimulated with 100 ng/ml DNP-HSA for 0, 2, 5, and 10 min, and lysates were cleared and immunoblotted for anti-phospho-p38 (Thr-180/182), and anti-total p38. A representative blot is shown above, and below are shown the average of three independent experiments and standard deviations for relative -fold induction over wild-type cells for phospho-p38 (p-p38)/total p38. (*, pvalue0.05,Student’sttest)B,degranulationofIgE-primedPak2fl/flandPak2/BMMCsstimulatedwith30ng/mlDNP-HSAantigenandincubatedwithp38 inhibitor SB203580 (10 M) 30 min prior to assay. Average -fold change of three independent experiments and standard deviations (*, p value 0.05, Student’s t test) are shown. C, ERK1/2 was probed on immunoblots from Pak2fl/fl and Pak2/ stimulated for 10 min with 100 ng/ml DNP-HSA antigen. A representative blot is shown, run for three independent experiments. pERK, phospho-ERK.

Article Snippet: Cre Activation—A retroviral vector for Cre recombinase (MSCV-CRE-ERT2) under tamoxifen control was used to excise Pak2 (Addgene plasmid 22776) (15).

Techniques: Activity Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

doi: 10.1074/jbc.m112.422295

Figure Lengend Snippet: FIGURE 6. Pak2 and MT protein phosphorylation. A, IgE-primed Pak2fl/fl and Pak2/ BMMCs were stimulated with 100 ng/ml DNP-HSA for 10 min, and lysates were cleared and immunoblotted for phospho-GEF-H1 (pGEFH1) (Ser-885), total GEF-H1, phospho-stathmin (pStathmin) (Ser-16), and actin (loading control). Representative blots are shown. B, left panel, the graph depicts the average of three independent experiments and standard deviations for the percentage of change in phospho-GEF-H1 (Ser-885), normalized to total GEF-H1 (*, p value 0.05, Student’s t test). Right panel, the graph depicts the average of three independent experiments and standard deviations for the percentage of change in phospho-stathmin (Ser-16) normalized to actin, relative to WT cells (*, p value 0.05, Student’s t test). C, representative blot of IgE-primed Pak1/ BMMCs stimulated with 100 ng/ml DNP-HSA for 10 min. Lysates were cleared and immunoblotted for phospho-GEFH1 (Ser-885), phospho-stathmin (Ser-16), and actin (loading control). D, the graph depicts -fold induction of phospho- GEFH1 and phospho-stathmin normalized to actin, average of three independent experiments, and standard deviations. E, in vitro kinase assay with recom- binant Pak2 and GEF-H1-GFP; lysates run on parallel membranes were probed with anti-phospho-GEF-H1 (Ser-885) and anti-GEF-H1.

Article Snippet: Cre Activation—A retroviral vector for Cre recombinase (MSCV-CRE-ERT2) under tamoxifen control was used to excise Pak2 (Addgene plasmid 22776) (15).

Techniques: Phospho-proteomics, Control, In Vitro, Kinase Assay

Journal: Journal of Biological Chemistry

Article Title: Pak2 Kinase Restrains Mast Cell FcϵRI Receptor Signaling through Modulation of Rho Protein Guanine Nucleotide Exchange Factor (GEF) Activity

doi: 10.1074/jbc.m112.422295

Figure Lengend Snippet: FIGURE 7. Model of Pak2 signaling in mast cells. A model demonstrating how Pak1 and Pak2 regulate IgE-mediated secretion in bone marrow-derived mast cells is shown. In this scenario, Pak1 and Pak2 play opposing roles. Pak1, which is present at lower levels than Pak2 in mast cells, acts through association with PP2A to promote ERM phosphorylation and augment secretion. Pak2 acts primarily through phosphorylation of GEF-H1 and stathmin/Op18 to limit RhoA activity, leading to diminished downstream activation of p38 and MLC2. Limiting the activity of these effectors results in diminished secretion. Differential spatial or temporal regulation of Pak1 versus Pak2 might determine the output (i.e. secretion or no secretion) under particular conditions. Ag, antigen; p, phosphorylation; MT, microtubule.

Article Snippet: Cre Activation—A retroviral vector for Cre recombinase (MSCV-CRE-ERT2) under tamoxifen control was used to excise Pak2 (Addgene plasmid 22776) (15).

Techniques: Derivative Assay, Phospho-proteomics, Activity Assay, Activation Assay