Journal: Molecular cancer research : MCR

Article Title: Heparan sulfate synthesized by Ext1 regulates receptor tyrosine kinase signaling and promotes resistance to EGFR inhibitors in GBM

doi: 10.1158/1541-7786.MCR-20-0420

Figure Lengend Snippet: (A) Representative images and relative quantification of total phosphotyrosines in HS-null and HS-wt cells (n=3 biologic replicates, mean ± SD). β-actin loading control. (B) Representative images of phosphorylation of Akt (S473), Akt (T308), and Erk1/2 (T202/T204) and total Akt and Erk1/2. Relative quantification of phosphorylation of Akt (S473) and Erk1/2 in HS-null and HS-wt cells (n=3 biologic replicates, mean ± SD). β-actin loading control. (C) Representative images and quantification of RTK phosphorylation (EGFR, ErbB2, FGFR3, FGFR4, InsulinR, IGF1R, Axl, PDGFRα and PDGFRβ) in HS-null and HS-wt cells as determined by phospho-RTK array. For all RTKs shown HS-null versus HS-wt was different by one-way ANOVA, p<0.001. (D) Tumor volume over time in mice harboring HS-null and HS-wt cells (n=5). (E) Proliferation, as determined by Ki-67, in tumor tissue (n=5 per group). (F) Relative vascular density in representative regions of HS-null and HS-wt tumors normalized to HS-wt (n=4 per group) (G,H) Representative images of HS expression (red) in HS-wt (G) and HS-null (H) tumors. Inset (h) demonstrates circumscribed tumor (#) in subcutaneous space (*) with modest HS expression (red). Nuclei are stained with DAPI. Scale bar 30μm. All data representative of at least two experiments, mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: Antibodies used for flow cytometry, Western blotting, and immunostaining included: mouse anti-heparan sulfate (clone 10E4, US Biological Cat #H1890, RRID:AB_10013601); mouse anti-heparan sulfate (clone F69–3G10, AMSBIO Cat# 370260–1, RRID:AB_10892311); mouse anti-chondroitin sulfate (clone CS-56, Sigma-Aldrich Cat #C8035, RRID:AB_476879); rabbit anti-Ki-67 (clone 30–9, Ventana Medical Systems Cat #790–4286, RRID:AB_2631262); mouse anti-phosphotyrosine (Millipore Cat #05–321, RRID:AB_309678), rabbit anti-phosphorylated PDGFRα (Y742) (R&D Systems Cat# AF2114, RRID:AB_416551); rabbit anti-PDGFRα (Millipore Cat #07–276, RRID:AB_310485); mouse anti-GAPDH antibody (Millipore Cat #MAB374, RRID:AB_2107445); and from Cell Signaling Technology rabbit anti-phosphorylated AKT (S473) (Cat #4060, RRID:AB_2315049), rabbit anti-AKT (Cat #9272, RRID:AB_329827), rabbit anti-phosphorylated ERK1/2 (T202/T204) (Cat #9101, RRID:AB_331646), mouse anti-ERK1/2 Cat #9107, RRID:AB_10695739), and rabbit anti-β-actin antibody (Cat #4967, RRID:AB_330288).

Techniques: Quantitative Proteomics, Control, Phospho-proteomics, Expressing, Staining

Journal: Molecular cancer research : MCR

Article Title: Heparan sulfate synthesized by Ext1 regulates receptor tyrosine kinase signaling and promotes resistance to EGFR inhibitors in GBM

doi: 10.1158/1541-7786.MCR-20-0420

Figure Lengend Snippet: (A) Quantification of cell surface HS in GBM43 with (ΔHS, black bar) and without (CNTL, clear bar) removal of cell surface HS by heparitinase I, II and III. Normalized median fluorescence intensity ± SD for three independent experiments. (B) Quantification of RTK phosphorylation (EGFR, InsulinR, IGF1R, and PDGFRα) in GBM43 with enzymatic HS removal (ΔHS) relative to control GBM43 (CNTL) as determined by phospho-RTK array. Representative of two independent experiments. (C,D) Representative flow cytometry for GBM43 and quantification of cell surface bound tagged PDGF-BB with (ΔHS, black line and black bars) or without (CNTL, grey line and white bars) removal of HS. Quantification is normalized median fluorescence intensity ± SD for three independent experiments. Data normalized to non-relevant IgM. (E) Representative Western blot demonstrating phosphorylation of PDGFRα (Y742), AKT (S473) and ERK1/2 (T202/T204) after PDGF-BB (100 ng) stimulation for 15 min with and without enzymatic removal of HS (H’ase). (F) Relative band intensities of p-PDGFRα (Y742), p-AKT (S473) and p-ERK1/2 (T202/T204) after normalization to total-PDGFRα, total-AKT and total-ERK1/2, respectively. *p < 0.05, **p < 0.01. GAPDH, loading control. (G) Exogenous heparin, a highly sulfated type of HS, promotes growth of patient-derived GBM43 in a dose-dependent manner. Cell viability determined in the presence of 10μg/mL or 100μg/mL heparin or 100μg/mL chondroitin sulfate A, or control (0, CNTL). Quantification is normalized cell viability ± SD for three independent experiments. (H) Representative Western blot demonstrating increased phosphorylation of PDGFRα (Y742) and ERK1/2 (T202/T204) after heparin treatment in GBM43.

Article Snippet: Antibodies used for flow cytometry, Western blotting, and immunostaining included: mouse anti-heparan sulfate (clone 10E4, US Biological Cat #H1890, RRID:AB_10013601); mouse anti-heparan sulfate (clone F69–3G10, AMSBIO Cat# 370260–1, RRID:AB_10892311); mouse anti-chondroitin sulfate (clone CS-56, Sigma-Aldrich Cat #C8035, RRID:AB_476879); rabbit anti-Ki-67 (clone 30–9, Ventana Medical Systems Cat #790–4286, RRID:AB_2631262); mouse anti-phosphotyrosine (Millipore Cat #05–321, RRID:AB_309678), rabbit anti-phosphorylated PDGFRα (Y742) (R&D Systems Cat# AF2114, RRID:AB_416551); rabbit anti-PDGFRα (Millipore Cat #07–276, RRID:AB_310485); mouse anti-GAPDH antibody (Millipore Cat #MAB374, RRID:AB_2107445); and from Cell Signaling Technology rabbit anti-phosphorylated AKT (S473) (Cat #4060, RRID:AB_2315049), rabbit anti-AKT (Cat #9272, RRID:AB_329827), rabbit anti-phosphorylated ERK1/2 (T202/T204) (Cat #9101, RRID:AB_331646), mouse anti-ERK1/2 Cat #9107, RRID:AB_10695739), and rabbit anti-β-actin antibody (Cat #4967, RRID:AB_330288).

Techniques: Fluorescence, Phospho-proteomics, Control, Flow Cytometry, Western Blot, Derivative Assay

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 3 Interaction of NHERF2 with ephrin-B1, nephrin, and podocalyxin. A: Interaction assays with rat glomerular lysates. A specific NHERF2 band of approximately 45 kDa was detected in the precipitate with antieephrin-B1 antibody (left panel), and with anti-nephrin antibody (right panel). B: Left panel: The assays with the human embryonic kidney (HEK) 293 cells cotransfected with NHERF2-strep Q21 and ephrin-B1eHA. The NHERF2 band was detected in the precipitate with anti-HA antibody. The ephrin-B1 band was detected in the precipitate with anti-strep antibody. Middle panel: The assay with the HEK293 cells transfected with NHERF2-strep and nephrin-FLAG. The nephrin band was not detected in the precipitate with anti-strep antibody. The NHERF2 band was not detected in the precipitate with anti-FLAG antibody. Right panel: The assay with the HEK293 cells transfected with NHERF2-strep, ephrin-B1eHA, and nephrin-FLAG. The NHERF2 band was detected in the precipitate with anti-FLAG antibody. The nephrin band was detected in the precipitate with anti-strep antibody. C: The NHERF2 band was not detected in the precipitate with anti-HA antibody in the HEK293 cells cotransfected with NHERF2-strep and the truncated ephrin-B1eHA in which PDZ binding motif was deleted. D: Left panel: The NHERF2 band was not detected in the precipitate with anti-HA antibody in the HEK cells transfected with ephrin-B1eHA and the truncated NHERF2-strep in which PDZ domain 1 was deleted. Right panel: The NHERF2 band was not detected in the precipitate with anti-myc antibody from the lysate of the HEK293 cells transfected with podocalyxin-myc and the truncated NHERF2-strep in which PDZ domain 2 was deleted. E: Left panel: The NHERF2 band in the precipitate with the antibody recognizing ephrin-B1 was diminished by the co- expression of podocalyxin. No interaction of ephrin-B1 with podocalyxin was detected. Right panel: The NHERF2 band in the precipitate with the anti- body recognizing podocalyxin was diminished by the co-expression of ephrin-B1. IP, immunoprecipitation; NMS, normal mouse serum; NRS, normal rabbit serum; WB, Western blot analysis.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Transfection, Binding Assay, Expressing, Immunoprecipitation, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 4 Nephrin interacts with ezrin via ephrin-B1 and NHERF2. A: Interaction assays with rat glomerular lysates. A specific ezrin band of approximately 80 kDa was detected in the precipitate with the anti-ephrin-B1 antibody (left panel), and in the precipitate with the anti-nephrin antibody (right panel). B: Left panel: An endogenous ezrin band was detected in the precipitate with the antibody recognizing ephrin-B1 in the HEK293 cells cotransfected with ephrin- B1 and NHERF2. The ezrin band was not detected in the precipitate with the antibody recognizing ephrin-B1 in the human embryonic kidney (HEK) 293 cells transfected with ephrin-B1 only. Right panel: The endogenous ezrin band was detected in the precipitate with the antibody recognizing NHERF2 in the HEK293 cells cotransfected with ephrin-B1 and NHERF2. The ezrin band was not detected in the precipitate with the antibody recognizing NHERF2 in the HEK293 cells transfected with NHERF2 only. Co-expressions of ephrin-B1 are required for the interaction of NHERF2 with ezrin. C: The assay with the HEK293 cells transfected with nephrin-FLAG, ephrin-B1eHA, and NHERF2-strep. The NHERF2 and ezrin bands were detected in the precipitate with the antibody recognizing nephrin from the lysate of the HEK293 cells transfected with nephrin, ephrin-B1, and NHERF2. Neither NHERF2 nor ezrin band was detected in the precipitates with the antibody detecting nephrin from the lysate of the HEK cells without transfection of ephrin-B1. IP, immunoprecipitation; NRS, normal rabbit serum; WB, Western blot analysis.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 10 Phosphorylation of ephrin-B1 disrupts the linkage of nephrineephrin-B1eNHERF2eezrin. A: The assay with the human embryonic kidney (HEK) 293 cells transfected with NHERF2-strep, ephrin-B1eHA, and nephrin-FLAG. Nephrin and ephrin-B1 were tyrosine phosphorylated by the anti-nephrin antibody treatment. The interactions of nephrineendogenous ezrin were detected in the HEK293 cells without the anti-nephrin antibody treatment. All of these in- teractions were disrupted in the HEK293 cells treated with the anti-nephrin antibody. B: The assay with the HEK293 cells transfected with NHERF2-strep and ephrin-B1eHA. Ephrin-B1 was phosphorylated by the EphB2-Fc treatment. The interactions of ephrin-B1eNHERF2, NHERF2eendogenous ezrin, and ephrin- B1eendogenous ezrin were detected in the HEK293 cells without Eph-Fc treatment. All of these interactions were disrupted in the HEK293 cells treated with the EphB2-Fc. NHERF2 and ezrin were serine/threonine phosphorylated in the HEK293 cells without the EphB2-Fc treatment. NHERF2 and ezrin were dephosphorylated by the EphB2-Fc treatment. IP, immunoprecipitation; WB, Western blot analysis Q22 .

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 11 Phosphorylation of ephrin-B1 disrupts the linkage of podocalyxin-NHERF2-ezrin. The assay with the human embryonic kidney (HEK) 293 cells transfected with NHERF2-strep, ephrin-B1eHA, and podocalyxin-myc. Ephrin-B1 was phosphorylated by the EphB2-Fc treatment. The interactions of podocalyxin-NHERF2, NHERF2eendogenous ezrin, and podocalyxineendogenous ezrin were detected in the HEK293 cells without the Eph-Fc treatment. The interactions of podocalyxin-NHERF2 and NHERF2eendogenous ezrin were disrupted in the HEK293 cells treated with EphB2-Fc. Left panel: The interaction of podocalyxineendogenous ezrin was still detected in the HEK293 cells treated with the EphB2-Fc. NHERF2 and ezrin were serine/threonine phosphorylated in the HEK293 cells without the EphB2-Fc treatment. Right panel: NHERF2 and ezrin were dephosphorylated by the EphB2-Fc treatment. IP, immunoprecipi- tation; WB, Western blot analysis Q23 .

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Phospho-proteomics, Transfection, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 1 NHERF2 expression is down-regulated in glomeruli of the ephrin-B1 conditional knockout (CKO) mice. A: Real-time PCR analysis of the isolated glomeruli of ephrin-B1 CKO mice. The glomerular mRNA expression of NHERF2 was decreased in the ephrin-B1 CKO mice (17.4% 5.7%; two-tailed t-test). Glomerular mRNA expression of ephrin-B1 was clearly decreased (0.86% 0.066%; two-tailed t-test). Glomerular mRNA expressions of zonula occludens protein 1 (ZO-1), MAGI-1, and CASK Q19 were not altered in the ephrin-B1 CKO mice. B: Representative immunofluorescence findings of NHERF2 and ephrin-B1 in kidney sections. The staining of NHERF2 was decreased in ephrin-B1 CKO mice (2.67 0.18 versus 3.59 0.16; U-test). No ephrin-B1 staining in glomeruli was detected Q20 . **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bar Z 20 mm (B). IF, immunofluorescence; WT, wild type.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Isolation, Two Tailed Test, Staining

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 2 Localization of NHERF2 in glomeruli. A: Representative immunofluorescence finding of NHERF2 in rat kidney section. NHERF2 staining was restrictedly detected in glomeruli in the kidney section, and NHERF2 staining in glomeruli was detected along the capillary loop as a podocyte pattern. B: Dual- labeling finding of NHERF2 (green) with glomerular cell markers (red). The staining of NHERF2 was clearly apart from Thy1.1, a mesangial cell marker, and rat endothelial cell antigen-1 (RECA-1), an endothelial cell marker. Some portions of the NHERF2 staining were costained with nephrin (arrow). Some portions of the NHERF2 staining were costained with ephrin-B1 (arrow), and almost all ephrin-B1 staining was costained with NHERF2. Some portions of the NHERF2 staining were costained with podocalyxin (arrow), but some other portions of NHERF2 were apart from podocalyxin (arrowhead). The ephrin-B1 staining was apart from podocalyxin. C: Immunoelectron microscopic finding of NHERF2. Gold particles for NHERF2 were detected not only at the apical area (arrowhead) but also at the slit diaphragm (arrows). D: Developmental expression of NHERF2 in glomeruli. NHERF2 staining was detected in presumptive podocytes of the early S-shaped body stage when ephrin-B1 staining appeared. At this stage, NHERF2 staining was detected along the whole cell surface, including the basal side. At the late S-shaped body stage, NHERF2 was detected along the whole cell surface, and NHERF2 at the basolateral side was costained with ephrin-B1. At the capillary and maturing stages, NHERF2 staining was detected at the lateral and apical areas, and the NHERF2 staining at the lateral area was costained with ephrin-B1 (arrows). Scale bars: 100 mm (A); 20 mm (B and D); 1 mm (C).

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Staining, Labeling, Marker, Expressing

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 3 Interaction of NHERF2 with ephrin-B1, nephrin, and podocalyxin. A: Interaction assays with rat glomerular lysates. A specific NHERF2 band of approximately 45 kDa was detected in the precipitate with antieephrin-B1 antibody (left panel), and with anti-nephrin antibody (right panel). B: Left panel: The assays with the human embryonic kidney (HEK) 293 cells cotransfected with NHERF2-strep Q21 and ephrin-B1eHA. The NHERF2 band was detected in the precipitate with anti-HA antibody. The ephrin-B1 band was detected in the precipitate with anti-strep antibody. Middle panel: The assay with the HEK293 cells transfected with NHERF2-strep and nephrin-FLAG. The nephrin band was not detected in the precipitate with anti-strep antibody. The NHERF2 band was not detected in the precipitate with anti-FLAG antibody. Right panel: The assay with the HEK293 cells transfected with NHERF2-strep, ephrin-B1eHA, and nephrin-FLAG. The NHERF2 band was detected in the precipitate with anti-FLAG antibody. The nephrin band was detected in the precipitate with anti-strep antibody. C: The NHERF2 band was not detected in the precipitate with anti-HA antibody in the HEK293 cells cotransfected with NHERF2-strep and the truncated ephrin-B1eHA in which PDZ binding motif was deleted. D: Left panel: The NHERF2 band was not detected in the precipitate with anti-HA antibody in the HEK cells transfected with ephrin-B1eHA and the truncated NHERF2-strep in which PDZ domain 1 was deleted. Right panel: The NHERF2 band was not detected in the precipitate with anti-myc antibody from the lysate of the HEK293 cells transfected with podocalyxin-myc and the truncated NHERF2-strep in which PDZ domain 2 was deleted. E: Left panel: The NHERF2 band in the precipitate with the antibody recognizing ephrin-B1 was diminished by the co- expression of podocalyxin. No interaction of ephrin-B1 with podocalyxin was detected. Right panel: The NHERF2 band in the precipitate with the anti- body recognizing podocalyxin was diminished by the co-expression of ephrin-B1. IP, immunoprecipitation; NMS, normal mouse serum; NRS, normal rabbit serum; WB, Western blot analysis.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Transfection, Binding Assay, Expressing, Immunoprecipitation, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 4 Nephrin interacts with ezrin via ephrin-B1 and NHERF2. A: Interaction assays with rat glomerular lysates. A specific ezrin band of approximately 80 kDa was detected in the precipitate with the anti-ephrin-B1 antibody (left panel), and in the precipitate with the anti-nephrin antibody (right panel). B: Left panel: An endogenous ezrin band was detected in the precipitate with the antibody recognizing ephrin-B1 in the HEK293 cells cotransfected with ephrin- B1 and NHERF2. The ezrin band was not detected in the precipitate with the antibody recognizing ephrin-B1 in the human embryonic kidney (HEK) 293 cells transfected with ephrin-B1 only. Right panel: The endogenous ezrin band was detected in the precipitate with the antibody recognizing NHERF2 in the HEK293 cells cotransfected with ephrin-B1 and NHERF2. The ezrin band was not detected in the precipitate with the antibody recognizing NHERF2 in the HEK293 cells transfected with NHERF2 only. Co-expressions of ephrin-B1 are required for the interaction of NHERF2 with ezrin. C: The assay with the HEK293 cells transfected with nephrin-FLAG, ephrin-B1eHA, and NHERF2-strep. The NHERF2 and ezrin bands were detected in the precipitate with the antibody recognizing nephrin from the lysate of the HEK293 cells transfected with nephrin, ephrin-B1, and NHERF2. Neither NHERF2 nor ezrin band was detected in the precipitates with the antibody detecting nephrin from the lysate of the HEK cells without transfection of ephrin-B1. IP, immunoprecipitation; NRS, normal rabbit serum; WB, Western blot analysis.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 5 Ezrin is dephosphorylated, and the F-actin structure is deranged, in NHERF2 knockdown podocytes. mRNA expression of NHERF2 decreased to 21.5% (two-tailed t-test), and the staining intensity of NHERF2 was reduced to 16.5% (two-tailed t-test) in the cells treated with siRNA for NHERF2 for 48 hours. Ephrin B1 staining was also clearly decreased (37.2% 2.46%; two-tailed t-test), and phosphorylated ezrin was decreased in the cells treated with the NHERF2 siRNA. Phosphorylation of ezrin was evaluated by the staining with the antiephosphorylated ezrin/moesin/radixin (ERM) antibody. The staining intensity of the phosphorylated ERM was decreased to 16.5% (two-tailed t-test), although the staining with the antibody detecting total ezrin was not clearly altered. The data are shown at the ratio (percentage) relative to the control siRNA-treated cell. F-actin was detected by rhodamine-phalloidin, and the phalloidin staining was evaluated as score 1 to 4. In the NHERF2 knockdown podocytes, cells with score 1 and 2 are fewer, and cells with score 3 and 4 are more than in the control podocytes (3.41 0.29 versus 2.06 0.54; NHERF2 siRNA versus control; U-test). *P < 0.05, **P < 0.01, and ****P < 0.0001. Scale bar Z 100 mm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Knockdown, Expressing, Two Tailed Test, Staining, Phospho-proteomics, Control

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 7 NHERF2 is dissociated from ezrin in the ephrin-B1 conditional knockout (CKO) mice. A: The expression and the phosphorylation of ezrin were decreased in the ephrin-B1 CKO mice (U-test). Stainings of nephrin and NHERF2 were decreased in the CKO mice, whereas the staining of podocalyxin was not altered in the CKO mice. B: Dual-labeling immunofluorescence (IF) findings of NHERF2 with ezrin and podocalyxin. NHERF2 was colocalized with ezrin almost completely in wild-type (WT) mice. In the CKO mice, NHERF2 was down-regulated and the remaining NHERF2 was clearly dissociated from ezrin (arrow). Some of NHERF2 was costained with podocalyxin in WT mice. The remaining NHERF2 was apart from podocalyxin in the CKO mice (arrow). C: Findings of the proximity ligation assay (PLA) technique. Almost no positive PLA signals showing close proximity of NHERF2 and podocalyxin were detected in the CKO mice (WT versus CKO, 28.54 1.40 versus 0.26 0.07; two-tailed t-test). **P < 0.01, ****P < 0.0001. Scale bar Z 20 mm (AeC). ERM, ezrin/moesin/radixin.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Knock-Out, Expressing, Phospho-proteomics, Staining, Labeling, Proximity Ligation Assay, Two Tailed Test

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 8 NHERF2 and phosphorylated ezrin are decreased in the rat nephrotic model induced by the anti-nephrin antibody injection. Down-regulation and tyrosine phosphorylation of ephrin-B1 were already detected at 1 hour after the disease induction. Stainings of NHERF2 and ezrin were decreased already at 1 hour. Podocalyxin staining was not altered through the period examined. Ezrin was threonine phosphorylated in normal rat glomeruli, and phosphorylated ezrin detected by the antiephosphorylated ezrin/moesin/radixin (ERM) was already decreased at 1 hour (2.28 0.09 versus 3.83 0.10; U-test). *P < 0.05. Scale bar Z 20 mm. IF, immunofluorescence.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Injection, Phospho-proteomics, Staining

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 9 The linkage of nephrineephrin-B1eNHERF2eezrin is disrupted in the anti-nephrin antibody-induced nephropathy. A: Dual-labeling immuno- fluorescence findings of NHERF2 with ephrin-B1, ezrin, and podocalyxin. Ephrin-B1 and nephrin were dissociated on day 5 when proteinuria peaked in this model. NHERF2 was almost completely colocalized with ezrin, and some portions of NHERF2 were colocalized with ephrin-B1 and other portions of NHERF2 were colocalized with podocalyxin in normal rat kidney sections. NHERF2 staining was decreased on day 5, and the remaining NHERF2 was isolated from ephrin-B1, ezrin, and podocalyxin (arrow). B: The findings of proximity ligation assay (PLA) technique. Almost no positive PLA signals showing close proximity of NHERF2 with ephrin-B1 or ezrin were detected on day 5 in this nephropathy (NHERF2/ephrin-B1: normal versus day 5: 89.22 4.75 versus 0.53 0.10, two-tailed t- test; NHERF2/ezrin: normal versus day 5: 142.23 8.96 versus 1.23 0.23, two-tailed t-test). ****P < 0.0001. Scale bar Z 20 mm (A and B).

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Labeling, Staining, Isolation, Proximity Ligation Assay, Two Tailed Test

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 10 Phosphorylation of ephrin-B1 disrupts the linkage of nephrineephrin-B1eNHERF2eezrin. A: The assay with the human embryonic kidney (HEK) 293 cells transfected with NHERF2-strep, ephrin-B1eHA, and nephrin-FLAG. Nephrin and ephrin-B1 were tyrosine phosphorylated by the anti-nephrin antibody treatment. The interactions of nephrineendogenous ezrin were detected in the HEK293 cells without the anti-nephrin antibody treatment. All of these in- teractions were disrupted in the HEK293 cells treated with the anti-nephrin antibody. B: The assay with the HEK293 cells transfected with NHERF2-strep and ephrin-B1eHA. Ephrin-B1 was phosphorylated by the EphB2-Fc treatment. The interactions of ephrin-B1eNHERF2, NHERF2eendogenous ezrin, and ephrin- B1eendogenous ezrin were detected in the HEK293 cells without Eph-Fc treatment. All of these interactions were disrupted in the HEK293 cells treated with the EphB2-Fc. NHERF2 and ezrin were serine/threonine phosphorylated in the HEK293 cells without the EphB2-Fc treatment. NHERF2 and ezrin were dephosphorylated by the EphB2-Fc treatment. IP, immunoprecipitation; WB, Western blot analysis Q22 .

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 11 Phosphorylation of ephrin-B1 disrupts the linkage of podocalyxin-NHERF2-ezrin. The assay with the human embryonic kidney (HEK) 293 cells transfected with NHERF2-strep, ephrin-B1eHA, and podocalyxin-myc. Ephrin-B1 was phosphorylated by the EphB2-Fc treatment. The interactions of podocalyxin-NHERF2, NHERF2eendogenous ezrin, and podocalyxineendogenous ezrin were detected in the HEK293 cells without the Eph-Fc treatment. The interactions of podocalyxin-NHERF2 and NHERF2eendogenous ezrin were disrupted in the HEK293 cells treated with EphB2-Fc. Left panel: The interaction of podocalyxineendogenous ezrin was still detected in the HEK293 cells treated with the EphB2-Fc. NHERF2 and ezrin were serine/threonine phosphorylated in the HEK293 cells without the EphB2-Fc treatment. Right panel: NHERF2 and ezrin were dephosphorylated by the EphB2-Fc treatment. IP, immunoprecipi- tation; WB, Western blot analysis Q23 .

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Phospho-proteomics, Transfection, Western Blot

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 12 Schematic diagram of the proposed pathogenic mechanism of disruption of the linkages of nephrineephrin-B1eNHERF2eezrin and podo- calyxineephrin-B1eNHERF2eezrin. A: The nephrineephrin-B1 complex at the slit diaphragm is linked to F-actin via NHERF2 and ezrin. Podocalyxin is an apical membrane protein linked to F-actin via NHERF2 and ezrin. Ephrin-B1 binds to the first PDZ domain of NHERF2, and podocalyxin binds to the second PDZ domain. But they do not share a molecule of NHREF2. It is estimated NHERF2 forms dimer. In the physiological state, nephrin and ephrin-B1 are not phosphorylated, and NHERF2 and ezrin are serine/threonine phosphorylated. B: If slit diaphragm is stimulated and nephrin and ephrin-B1 are tyrosine phosphorylated, nephrin is dissociated from ephrin-B1, and ephrin-B1 is dissociated from NHERF2. Then, NHERF2 changed to be an inactive dephosphorylated form and then dissociated from ezrin. Ezrin, which is dissociated from NHERF2, changed to be an inactive dephosphorylated form unable to bind F-actin. The phosphorylation of ephrin-B1 also dephosphorylated a neighboring NHERF2 of the linkage of podocalyxin-NHERF2-ezrin and disrupted the linkage Q24 .

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Disruption, Membrane, Phospho-proteomics

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 5 Ezrin is dephosphorylated, and the F-actin structure is deranged, in NHERF2 knockdown podocytes. mRNA expression of NHERF2 decreased to 21.5% (two-tailed t-test), and the staining intensity of NHERF2 was reduced to 16.5% (two-tailed t-test) in the cells treated with siRNA for NHERF2 for 48 hours. Ephrin B1 staining was also clearly decreased (37.2% 2.46%; two-tailed t-test), and phosphorylated ezrin was decreased in the cells treated with the NHERF2 siRNA. Phosphorylation of ezrin was evaluated by the staining with the antiephosphorylated ezrin/moesin/radixin (ERM) antibody. The staining intensity of the phosphorylated ERM was decreased to 16.5% (two-tailed t-test), although the staining with the antibody detecting total ezrin was not clearly altered. The data are shown at the ratio (percentage) relative to the control siRNA-treated cell. F-actin was detected by rhodamine-phalloidin, and the phalloidin staining was evaluated as score 1 to 4. In the NHERF2 knockdown podocytes, cells with score 1 and 2 are fewer, and cells with score 3 and 4 are more than in the control podocytes (3.41 0.29 versus 2.06 0.54; NHERF2 siRNA versus control; U-test). *P < 0.05, **P < 0.01, and ****P < 0.0001. Scale bar Z 100 mm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Knockdown, Expressing, Two Tailed Test, Staining, Phospho-proteomics, Control

Journal: The American journal of pathology

Article Title: Nephrin-Ephrin-B1-Na + /H + Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

doi: 10.1016/j.ajpath.2021.04.004

Figure Lengend Snippet: Figure 8 NHERF2 and phosphorylated ezrin are decreased in the rat nephrotic model induced by the anti-nephrin antibody injection. Down-regulation and tyrosine phosphorylation of ephrin-B1 were already detected at 1 hour after the disease induction. Stainings of NHERF2 and ezrin were decreased already at 1 hour. Podocalyxin staining was not altered through the period examined. Ezrin was threonine phosphorylated in normal rat glomeruli, and phosphorylated ezrin detected by the antiephosphorylated ezrin/moesin/radixin (ERM) was already decreased at 1 hour (2.28 0.09 versus 3.83 0.10; U-test). *P < 0.05. Scale bar Z 20 mm. IF, immunofluorescence.

Article Snippet: HEK293 Cell Transfection Assays with human embryonic kidney (HEK) 293 cell transfection were performed, as previously described.12,16 HEK293 cells were transfected with TwinStrepII-tagged SLC9A3R2 (NHERF2; Addgene, Cambridge, MA; plasmid number 83,200), myc-DDK-tagged podocalyxin (OriGene Technologies, Rockville, MD; catalog number RR213127), HA-tagged ephrin-B1,12 or FLAG-tagged nephrin12 by calcium phosphate method Q. ajp.amjpathol.org - The American Journal of Pathology 248 5 May 2021 6:26 pm EO: AJPA-D-20-00617 Q14 Q15 16 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 Immunoprecipitation Assays with Glomerular Lysates and Transfected HEK293 Cell Lysates Immunoprecipitation assays with rat glomerular lysates and transfected HEK293 cell lysates were performed basically, according to the method reported previously11,12,16 with anti-NHERF2 antibody (Santa Cruz Biotechnology), antieephrin-B1 antibody (Santa Cruz Biotechnology), antinephrin antibody,22 anti-ezrin antibody (Invitrogen), rabbit anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-HA antibody (Bethyl Laboratories), mouse anti-HA antibody (Santa Cruz Biotechnology), rabbit anti-myc antibody (MBL, Woburn, MA), mouse anti-myc antibody (Santa Cruz Biotechnology), mouse anti-strep antibody (MBL), rabbit antiephosphorylated ephrin-B (Cell Signaling), rabbit antiephosphorylated nephrin (Abcam), mouse antiephosphorylated serine antibody (SigmaAldrich), mouse antiephosphorylated threonine antibody (Cell Signaling), or mouse antiephosphorylated tyrosine antibody (Merk, Darmstadt, Germany).

Techniques: Injection, Phospho-proteomics, Staining

Journal: The American journal of pathology

Article Title: Murine Double Minute-2 Inhibition Ameliorates Established Crescentic Glomerulonephritis.

doi: 10.1016/j.ajpath.2016.01.017

Figure Lengend Snippet: Figure 1 Renal Q13 MDM2 expression. A: The expression of MDM2, p53, phosphorylated-MDM2, and phosphorylated-p53 in kidney of antiserum-induced GN mouse model and control animals was determined by Western blot analysis. b-Actin staining is shown as a loading control. The graph on the right shows the quantification of several Western blot analyses. B and C: MDM2 immunostaining (red) was performed on paraffin-embedded kidney samples of antiserum- induced GN mouse model and control adult C57BL/6 mice. B: Colocalization of MDM2 (arrows) with claudin (green) that stains the PECs. In antiserum- induced GN glomeruli MDM2 is strongly expressed in activated PECs (arrows). C: MDM2 (red) colocalization with CD31 stained glomerular endothelial cells (green). MDM2 expression is diminished in glomerular endothelial cells in antiserum-induced GN glomeruli. TO-PRO-3 stains nuclei in blue. *P < 0.05, **P < 0.01 versus baseline. Original magnification, 630. CTRL, control; GBM, glomerular basement membrane; GN, glomerulonephritis; MDM2, murine double minute-2; PEC, parietal epithelial cell.

Article Snippet: Protein Isolation and Western Blot Analysis We extracted protein from kidney tissues with the use of RIPA buffer (Sigma-Aldrich, Hamburg, Germany) that contained protease inhibitors (Roche) and processed it for Western blot ajp.amjpathol.org - The American Journal of Pathology 248 f 22 April 2016 8:46 pm EO: AJP15_0455 9 Table 1 Primers Used for Real-Time PCR Gene Forward sequence Reverse sequence MDM2 50-TGTGAAGGAGCACAGGAAAA-30 50-TCCTTCAGATCACTCCCACC-30 p53 50-CTAGCATTCAGGCCCCTCATC-30 50-TCCGACTGTGACTCCTCCAT-30 Ccl5 50-GTGCCCACGTCAAGGAGTAT-30 50-CCACTTCTTCTCTGGGTTGG-30 Il6 50-TGATGCACTTGCAGAAAACA-30 50-ACCAGAGGAAATTTTCAATAGGC-30 iNOS 50-TGAAGAAAACCCCTTGTGCT-30 50-TTCTGTGCTGTCCCAGTGAG-30 Nphs2 50-CAGGAAGCAGATGTCCCAGT-30 50-TGACGTTCCCTTTTTCCATC-30 Nphs1 50-CTCTTTCTACCGCCTCAACG-30 50-TTAGCAGACACGGACACAGG-30 Synpo 50-GCCAGGGACCAGCCAGATA-30 50-AGGAGCCCAGGCCTTCTCT-30 CD2AP 50-CATCCCTCGTCTCCCATTTA-30 50-TATCCGAGTTGGGGAAATCA-30 IL10 50-TGTCAAATTCATTCATGGCCT-30 50-ATCGATTTCTCCCCTGTGAA-30 IFNG 50-TGAGCTCATTGAATGCTTGG-30 50-ACAGCAAGGCGAAAAAGGAT-30 Il12a 50-GCTTCTCCCACAGGAGGTTT-30 50-CTAGACAAGGGCATGCTGGT-30 Il12b 50-AGTCCCTTTGGTCCAGTGTG-30 50-AGCAGTAGCAGTTCCCCTGA-30 MR1 50-GTGGATTGTCTTGTGGAGCA-30 50-TTGTGGTGAGCTGAAAGGTG-30 YM1 50-TTTCTCCAGTGTAGCCATCCTT-30 50-TCTGGGTACAAGATCCCTGAA-30 Fizz 50-CTGGATTGGCAAGAAGTTCC-30 50-CCCTTCTCATCTGCATCTCC-30 Arg1 50-TTTTTCCAGCAGACCAGCTT-30 50-AGAGATTATCGGAGCGCCTT-30 18s 50-GCAATTATTCCCCATGAACG-30 50-AGGGCCTCACTAAACCATCC-30 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 analysis as described.7 The membranes were incubated overnight at 4 C with primary antibodies, goat antibody against mouse p53 (Santa Cruz Biotechnology), rabbit antibody against MDM-2 (Abcam), phosphorylated-53 (Santa Cruz Biotechnology), phosphorylated-MDM2 (Cell Signaling Technology Inc., Danvers, MA), and rabbit antibody against b-actin (Cell Signaling Technology Inc.).

Techniques: Expressing, Control, Western Blot, Staining, Immunostaining, Membrane

Journal: The American journal of pathology

Article Title: Murine Double Minute-2 Inhibition Ameliorates Established Crescentic Glomerulonephritis.

doi: 10.1016/j.ajpath.2016.01.017

Figure Lengend Snippet: Figure 3 Nutlin-3a treatment suppresses most but not all of the pathologic features of crescentic GN in p53-deficient mice. A: Urine albumin/ creatinine ratio. B: BUN concentrations were determined 7 days after sheep GBM antiserum in- jection in p53-deficient mice. C: Glomerular injury was quantified on PAS-stained renal sections as described in Materials and Methods. D: Represen- tative images of kidney sections stained for CD31 as a marker of glomerular capillaries and a quan- tification of mean fluorescence area of CD31 positivity in glomeruli. E: Representative images of WT-1/nephrin costaining that was used to quantify differentiated podocytes in kidneys of vehicle- and nutlin-3aetreated kidneys. Numbers of nephrin/ WT-1 double-positive cells were quantified in both groups at 7 days after induction of anti-GBM disease. F: Representative images of PAS staining showing amelioration of crescent formation and glomerulosclerosis in nutlin-3aetreated mice compared with control group. Quantification of crescents from sections stained with PAS taken at day 7. Data are expressed as means SD. n Z 5 mice in each group. *P < 0.05, ***P < 0.005, nutlin-3ae versus vehicle-treated mice. Original magnification Q15 : 630; 400. BUN, blood urea nitrogen; GBM, glomerular basement membrane; GN, glomerulonephritis; PAS, periodic acid-Schiff.

Article Snippet: Protein Isolation and Western Blot Analysis We extracted protein from kidney tissues with the use of RIPA buffer (Sigma-Aldrich, Hamburg, Germany) that contained protease inhibitors (Roche) and processed it for Western blot ajp.amjpathol.org - The American Journal of Pathology 248 f 22 April 2016 8:46 pm EO: AJP15_0455 9 Table 1 Primers Used for Real-Time PCR Gene Forward sequence Reverse sequence MDM2 50-TGTGAAGGAGCACAGGAAAA-30 50-TCCTTCAGATCACTCCCACC-30 p53 50-CTAGCATTCAGGCCCCTCATC-30 50-TCCGACTGTGACTCCTCCAT-30 Ccl5 50-GTGCCCACGTCAAGGAGTAT-30 50-CCACTTCTTCTCTGGGTTGG-30 Il6 50-TGATGCACTTGCAGAAAACA-30 50-ACCAGAGGAAATTTTCAATAGGC-30 iNOS 50-TGAAGAAAACCCCTTGTGCT-30 50-TTCTGTGCTGTCCCAGTGAG-30 Nphs2 50-CAGGAAGCAGATGTCCCAGT-30 50-TGACGTTCCCTTTTTCCATC-30 Nphs1 50-CTCTTTCTACCGCCTCAACG-30 50-TTAGCAGACACGGACACAGG-30 Synpo 50-GCCAGGGACCAGCCAGATA-30 50-AGGAGCCCAGGCCTTCTCT-30 CD2AP 50-CATCCCTCGTCTCCCATTTA-30 50-TATCCGAGTTGGGGAAATCA-30 IL10 50-TGTCAAATTCATTCATGGCCT-30 50-ATCGATTTCTCCCCTGTGAA-30 IFNG 50-TGAGCTCATTGAATGCTTGG-30 50-ACAGCAAGGCGAAAAAGGAT-30 Il12a 50-GCTTCTCCCACAGGAGGTTT-30 50-CTAGACAAGGGCATGCTGGT-30 Il12b 50-AGTCCCTTTGGTCCAGTGTG-30 50-AGCAGTAGCAGTTCCCCTGA-30 MR1 50-GTGGATTGTCTTGTGGAGCA-30 50-TTGTGGTGAGCTGAAAGGTG-30 YM1 50-TTTCTCCAGTGTAGCCATCCTT-30 50-TCTGGGTACAAGATCCCTGAA-30 Fizz 50-CTGGATTGGCAAGAAGTTCC-30 50-CCCTTCTCATCTGCATCTCC-30 Arg1 50-TTTTTCCAGCAGACCAGCTT-30 50-AGAGATTATCGGAGCGCCTT-30 18s 50-GCAATTATTCCCCATGAACG-30 50-AGGGCCTCACTAAACCATCC-30 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 analysis as described.7 The membranes were incubated overnight at 4 C with primary antibodies, goat antibody against mouse p53 (Santa Cruz Biotechnology), rabbit antibody against MDM-2 (Abcam), phosphorylated-53 (Santa Cruz Biotechnology), phosphorylated-MDM2 (Cell Signaling Technology Inc., Danvers, MA), and rabbit antibody against b-actin (Cell Signaling Technology Inc.).

Techniques: Staining, Marker, Control, Membrane

Journal: The American journal of pathology

Article Title: Murine Double Minute-2 Inhibition Ameliorates Established Crescentic Glomerulonephritis.

doi: 10.1016/j.ajpath.2016.01.017

Figure Lengend Snippet: Figure 4 MDM2 Q16 blockade suppresses expression of inflammatory mediators and recruitment of leukocytes and macrophages in crescentic GN in wild-type and in p53-deficient mice. AeC: Antiserum-induced GN in wild-type C57BL/6 mice model. A: Total mRNA was prepared from vehicle- and nutlin-3aetreated kidneys. The mRNA levels were determined for the indicated cytokines and chemokines by real-time PCR and expressed as a mean of the ratio versus the respective 18s rRNA level. B: Representative images of kidney sections from vehicle- and nutlin-3aetreated kidneys stained for neutrophil and macrophages markers. C: Glomerular neutrophil (Ly-6B.2) and macrophage (mac-2) infiltrates are quantified by counting positive cells per glomerulus. DeF: Antiserum- induced GN in p53-deficient mice model. D: mRNA expression of inflammatory mediators in vehicle- and nutlin-3aetreated kidneys. E: Representative images of kidney sections from vehicle- and nutlin-3aetreated kidneys stained for neutrophil and macrophages markers. F: Glomerular neutrophil (Ly-6B.2) and macrophage (mac-2) infiltrates are quantified by counting positive cells per glomerulus. Data are expressed as means SD. *P < 0.05, **P < 0.01, and ***P < 0.005 nutlin-3a versus vehicle group. CCL5, chemokine ligand 5; GN, glomerulonephritis; iNOS, inducible nitric oxide synthase; MDM2, murine double minute-2; RANTES, regulated on activation normal T cell expressed and secreted.

Article Snippet: Protein Isolation and Western Blot Analysis We extracted protein from kidney tissues with the use of RIPA buffer (Sigma-Aldrich, Hamburg, Germany) that contained protease inhibitors (Roche) and processed it for Western blot ajp.amjpathol.org - The American Journal of Pathology 248 f 22 April 2016 8:46 pm EO: AJP15_0455 9 Table 1 Primers Used for Real-Time PCR Gene Forward sequence Reverse sequence MDM2 50-TGTGAAGGAGCACAGGAAAA-30 50-TCCTTCAGATCACTCCCACC-30 p53 50-CTAGCATTCAGGCCCCTCATC-30 50-TCCGACTGTGACTCCTCCAT-30 Ccl5 50-GTGCCCACGTCAAGGAGTAT-30 50-CCACTTCTTCTCTGGGTTGG-30 Il6 50-TGATGCACTTGCAGAAAACA-30 50-ACCAGAGGAAATTTTCAATAGGC-30 iNOS 50-TGAAGAAAACCCCTTGTGCT-30 50-TTCTGTGCTGTCCCAGTGAG-30 Nphs2 50-CAGGAAGCAGATGTCCCAGT-30 50-TGACGTTCCCTTTTTCCATC-30 Nphs1 50-CTCTTTCTACCGCCTCAACG-30 50-TTAGCAGACACGGACACAGG-30 Synpo 50-GCCAGGGACCAGCCAGATA-30 50-AGGAGCCCAGGCCTTCTCT-30 CD2AP 50-CATCCCTCGTCTCCCATTTA-30 50-TATCCGAGTTGGGGAAATCA-30 IL10 50-TGTCAAATTCATTCATGGCCT-30 50-ATCGATTTCTCCCCTGTGAA-30 IFNG 50-TGAGCTCATTGAATGCTTGG-30 50-ACAGCAAGGCGAAAAAGGAT-30 Il12a 50-GCTTCTCCCACAGGAGGTTT-30 50-CTAGACAAGGGCATGCTGGT-30 Il12b 50-AGTCCCTTTGGTCCAGTGTG-30 50-AGCAGTAGCAGTTCCCCTGA-30 MR1 50-GTGGATTGTCTTGTGGAGCA-30 50-TTGTGGTGAGCTGAAAGGTG-30 YM1 50-TTTCTCCAGTGTAGCCATCCTT-30 50-TCTGGGTACAAGATCCCTGAA-30 Fizz 50-CTGGATTGGCAAGAAGTTCC-30 50-CCCTTCTCATCTGCATCTCC-30 Arg1 50-TTTTTCCAGCAGACCAGCTT-30 50-AGAGATTATCGGAGCGCCTT-30 18s 50-GCAATTATTCCCCATGAACG-30 50-AGGGCCTCACTAAACCATCC-30 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 analysis as described.7 The membranes were incubated overnight at 4 C with primary antibodies, goat antibody against mouse p53 (Santa Cruz Biotechnology), rabbit antibody against MDM-2 (Abcam), phosphorylated-53 (Santa Cruz Biotechnology), phosphorylated-MDM2 (Cell Signaling Technology Inc., Danvers, MA), and rabbit antibody against b-actin (Cell Signaling Technology Inc.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Activation Assay

Journal: The American journal of pathology

Article Title: Murine Double Minute-2 Inhibition Ameliorates Established Crescentic Glomerulonephritis.

doi: 10.1016/j.ajpath.2016.01.017

Figure Lengend Snippet: Figure 5 MDM2 inhibition in vitro suppresses inflammation in glomerular endothelial cells and proliferation of PECs. A: Total mRNA was prepared from GEnCs transfected with MDM2 siRNA and control siRNA and treated with LPS or vehicle. The mRNA levels were determined for the indicated cytokines and chemokines by real-time PCR and expressed as a mean of the ratio versus the respective 18s rRNA level. B: PECs were grown in 0% or 20% serum-containing medium to confirm the proliferatory effect of serum on PECs. The proliferating PECs were then treated with increasing concentrations of MDM2 inhibitor Nut. Cell viability/ proliferation was determined after 24 hours by MTT assay. C: PECs were transfected with p53 siRNA and control siRNA and treated with increasing dose of Nut. The MTT assay shows complete reversal of Nut effect by p53 depletion in PECs. The graph below shows the efficiency of the p53 knockdown. All experiments were performed in triplicates. Data are expressed as means SD. *P < 0.05, **P < 0.01, and ***P < 0.005. CCL5, chemokine ligand 5; Ctrl, control; FBS, fetal bovine serum; GEnC, glomerular endothelial cell; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; Nut, nutlin-3a; PEC, parietal epithelial cell.

Article Snippet: Protein Isolation and Western Blot Analysis We extracted protein from kidney tissues with the use of RIPA buffer (Sigma-Aldrich, Hamburg, Germany) that contained protease inhibitors (Roche) and processed it for Western blot ajp.amjpathol.org - The American Journal of Pathology 248 f 22 April 2016 8:46 pm EO: AJP15_0455 9 Table 1 Primers Used for Real-Time PCR Gene Forward sequence Reverse sequence MDM2 50-TGTGAAGGAGCACAGGAAAA-30 50-TCCTTCAGATCACTCCCACC-30 p53 50-CTAGCATTCAGGCCCCTCATC-30 50-TCCGACTGTGACTCCTCCAT-30 Ccl5 50-GTGCCCACGTCAAGGAGTAT-30 50-CCACTTCTTCTCTGGGTTGG-30 Il6 50-TGATGCACTTGCAGAAAACA-30 50-ACCAGAGGAAATTTTCAATAGGC-30 iNOS 50-TGAAGAAAACCCCTTGTGCT-30 50-TTCTGTGCTGTCCCAGTGAG-30 Nphs2 50-CAGGAAGCAGATGTCCCAGT-30 50-TGACGTTCCCTTTTTCCATC-30 Nphs1 50-CTCTTTCTACCGCCTCAACG-30 50-TTAGCAGACACGGACACAGG-30 Synpo 50-GCCAGGGACCAGCCAGATA-30 50-AGGAGCCCAGGCCTTCTCT-30 CD2AP 50-CATCCCTCGTCTCCCATTTA-30 50-TATCCGAGTTGGGGAAATCA-30 IL10 50-TGTCAAATTCATTCATGGCCT-30 50-ATCGATTTCTCCCCTGTGAA-30 IFNG 50-TGAGCTCATTGAATGCTTGG-30 50-ACAGCAAGGCGAAAAAGGAT-30 Il12a 50-GCTTCTCCCACAGGAGGTTT-30 50-CTAGACAAGGGCATGCTGGT-30 Il12b 50-AGTCCCTTTGGTCCAGTGTG-30 50-AGCAGTAGCAGTTCCCCTGA-30 MR1 50-GTGGATTGTCTTGTGGAGCA-30 50-TTGTGGTGAGCTGAAAGGTG-30 YM1 50-TTTCTCCAGTGTAGCCATCCTT-30 50-TCTGGGTACAAGATCCCTGAA-30 Fizz 50-CTGGATTGGCAAGAAGTTCC-30 50-CCCTTCTCATCTGCATCTCC-30 Arg1 50-TTTTTCCAGCAGACCAGCTT-30 50-AGAGATTATCGGAGCGCCTT-30 18s 50-GCAATTATTCCCCATGAACG-30 50-AGGGCCTCACTAAACCATCC-30 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 analysis as described.7 The membranes were incubated overnight at 4 C with primary antibodies, goat antibody against mouse p53 (Santa Cruz Biotechnology), rabbit antibody against MDM-2 (Abcam), phosphorylated-53 (Santa Cruz Biotechnology), phosphorylated-MDM2 (Cell Signaling Technology Inc., Danvers, MA), and rabbit antibody against b-actin (Cell Signaling Technology Inc.).

Techniques: Inhibition, In Vitro, Transfection, Control, Real-time Polymerase Chain Reaction, MTT Assay, Knockdown