Journal: Human Reproduction (Oxford, England)

Article Title: Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation

doi: 10.1093/humrep/deab032

Figure Lengend Snippet: Podocalyxin (PCX) overexpression in Ishikawa cells. PCX was stably overexpressed (PCX-OE) in Ishikawa cells and compared with control (CON) cells. (A) Real-time RT-PCR analysis of PCX mRNA in CON and PCX-OE cells. Data normalized to 18S rRNA and expressed as fold change relative to CON (n = 4, *P < 0.05). (B) Representative western blot analysis of PCX protein, β-actin used as loading control. (C) Representative immunofluorescence analysis of PCX, F-actin and polarity marker scribble in control and PCX-OE cells. Inserts, negative controls. Scale bars, 20 µm.

Article Snippet: Stable overexpression of PCX in Ishikawa cells An expression construct of human PCX open reading frame (RC210816) and the empty pCMV6 vector (control plasmid) were purchased from Origene (Rockville, USA).

Techniques: Over Expression, Stable Transfection, Control, Quantitative RT-PCR, Western Blot, Immunofluorescence, Marker

Journal: Human Reproduction (Oxford, England)

Article Title: Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation

doi: 10.1093/humrep/deab032

Figure Lengend Snippet: Podocalyxin (PCX) inhibition of primary human trophoblast spheroid attachment. Equal numbers of trophoblast spheroids were co-cultured on top of control (CON) or PCX-overexpressing (PCX-OE) Ishikawa monolayer, and the percentage of spheroid attachment (number of attached/added) was examined over 24 h. Data expressed as median with interquartile range (n = 3-5). *P < 0.05 and **P < 0.01.

Article Snippet: Stable overexpression of PCX in Ishikawa cells An expression construct of human PCX open reading frame (RC210816) and the empty pCMV6 vector (control plasmid) were purchased from Origene (Rockville, USA).

Techniques: Inhibition, Cell Culture, Control

Journal: Human Reproduction (Oxford, England)

Article Title: Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation

doi: 10.1093/humrep/deab032

Figure Lengend Snippet: Podocalyxin (PCX) inhibition of primary human trophoblast spheroid invasion. Trophoblast spheroid invasion through control (CON) or PCX-overexpressing (PCX-OE) Ishikawa monolayer was examined at 24 h and 48 h. (A) Representative confocal images of side- and bottom-views of trophoblast spheroids (green) and Ishikawa monolayer (red). (B) Quantification of spheroid invasion. The volume of spheroid present beneath the Ishikawa monolayer was quantified by surface mapping. Data expressed as median with interquartile range (n = 4 for 24 h, n = 6 for 48 h). *P < 0.05.

Article Snippet: Stable overexpression of PCX in Ishikawa cells An expression construct of human PCX open reading frame (RC210816) and the empty pCMV6 vector (control plasmid) were purchased from Origene (Rockville, USA).

Techniques: Inhibition, Control

Journal: Science advances

Article Title: Spatially resolved cell polarity proteomics of a human epiblast model.

doi: 10.1126/sciadv.abd8407

Figure Lengend Snippet: Fig. 1. APEX2-based spatial biotinylation of apical and basolateral membrane territories in an hPSC-cyst epiblast model. (A) Design of APEX2 fusion construct based on a DOX-inducible piggyBac transposon system. GoI, gene of interest; TR, terminal repeats; TRE, tetracycline-responsive element; pA, polyadenylation sequence; rEf1, rat elongation factor 1 promoter; rtTA, reverse tetracycline transactivator; IRES, internal ribosome entry site; Puro, puromycin. (B) Flow diagram of APEX2-based proximity biotinylation of apical, basolateral, and cytoplasmic domains using APEX2 fusion proteins (e.g., PODXL, ATP1B1, or NES). hPSC-cysts expressing each construct were incubated with BP (biotin-phenol), followed by a 1.5-min H2O2 treatment to trigger biotinylation: APEX2 converts BP to biotin-phenoxyl radicals, which form covalent bonds with proximal endogenous proteins. The red stripe indicates the labeling radius of APEX2 (~20 nm). (C) hPSC-cyst APEX2 labeling assay timeline. (D) Optical section of indicated d3 APEX2 hPSC-cysts stained with indicated markers. FLAG (red) indicates the APEX2 construct localization. (E) Streptavidin (SA-555) staining of d3 APEX2 hPSC-cysts after labeling, showing spatially distinct streptavidin signals. White arrowhead indicates biotinylated intracellular vesicles. (F) Western blot of whole-cell lysates from d3 hPSC-cysts using indicated conditions was probed for biotin, revealing distinct patterns of construct-specific biotinylation. Red arrowheads indicate unique bands. Right: Total protein staining. (G) Coomassie staining following enrichment of biotinylated proteins from indicated samples. Some background biotinyla- tion signal is seen in unlabeled (2, 4, 6, 8, and 10) and negative control (11 and 12) samples. Scale bars, 20 m unless otherwise noted. Nuclei: Hoechst (blue).

Article Snippet: A DOX-inducible piggyBac transposon system (PB-TA-ERP2; gift of K. Woltjen; Addgene, #80477) (81) was used to generate five stable transgenic H9 hESC lines expressing APEX2 fused to human PODXL (OriGene; NM_00108111), EZR (gift of D. Louvard, Curie Institute, Paris), ATP1B1 (GenScript; NM_001677.3), SDC1 (GenScript; NM_002997.4), or NES (gift of A. Ting, Stanford; Addgene, #49386) (Fig. 1A) (82).

Techniques: Membrane, Construct, Sequencing, Expressing, Incubation, Labeling, Staining, Western Blot, Negative Control

Journal: Science advances

Article Title: Spatially resolved cell polarity proteomics of a human epiblast model.

doi: 10.1126/sciadv.abd8407

Figure Lengend Snippet: Fig. 2. Ratiometric profiling of cell polarity proteomes in hPSC-cysts. (A) Workflow of hPSC-cyst cell polarity proteome analysis, using three replicates of apical (2× PODXL and 1× EZR), basolateral (2× ATP1B1 and 1× SDC1), and 3× cytoplasmic (NES) samples and one negative control (NES without H2O2). (B) Experimental design of 10-plex TMT-based profiling. Row 1, specific TMT tags; row 2, APEX2 lines; rows 3 and 4, proximity labeling conditions. POI, protein of interest. (C) Flow diagram of ratiometric analyses to obtain proteins specific to apical and basolateral territories. Brackets: Number of proteins that passed each filter. (D and E) Representative histograms and receiver operating characteristic (ROC) curves of apical replicate 1 (D) and basolateral replicate 1 (E), illustrating how filters 1, 2, and 3 were applied. See fig. S2 (A to D) and Materials and Methods for additional cutoff analysis details. (F) Representative scatterplots (apical replicates 1 and 2) revealing strong correlation among biological replicates (additional plots in fig. S2E). (G and H) Representative ratiometric analysis scatter plots [(G), apical territory replicate #1 (PODXL-APEX2, TMT10-126); (H), basolateral replicate #1 (APEX2-ATP1B1, TMT10-128N)] showing the distribution of proteins based on TMT ratios [x axis, 126 or 128N/negative (131); y axis, 126 or 128N/129C (cytoplasmic #1)]. Orange shade (top right quadrant) indicates proteins that were analyzed for filter 3. Colored dots/fonts indicate proteins with existing apical (red, top)/basolateral (red, bottom), or plasma membrane and cell membrane (green) annotations; others in gray. See fig. S2H for additional plots. (I) Representative ratiometric scatter plots based on normalized TMT ratios of apical #1 (x axis, 126/131) and basolateral #1 (128N/131) of 4454 proteins revealing a clear enrichment of proteins from apical (red), basolateral (blue), and nonpolar (green) lists in distinct domains of the scatter plot. Proteins identified through filter 4 are indicated in gray fonts. See fig. S2I for additional plots.

Article Snippet: A DOX-inducible piggyBac transposon system (PB-TA-ERP2; gift of K. Woltjen; Addgene, #80477) (81) was used to generate five stable transgenic H9 hESC lines expressing APEX2 fused to human PODXL (OriGene; NM_00108111), EZR (gift of D. Louvard, Curie Institute, Paris), ATP1B1 (GenScript; NM_001677.3), SDC1 (GenScript; NM_002997.4), or NES (gift of A. Ting, Stanford; Addgene, #49386) (Fig. 1A) (82).

Techniques: Negative Control, Labeling, Clinical Proteomics, Membrane

Journal: Kidney international

Article Title: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis

doi: 10.1038/ki.2013.354

Figure Lengend Snippet: Pedigree of family FG-HI, sequencing and multisequence alignment. (a) Pedigree for family FG-HI. Affected individuals are indicated in grey. Individuals who are heterozygous for the variant PODXL p.L442R are denoted by “+” while those without the mutation are denoted by “−”. Individuals without a notation were not tested because no sample was available. (b) Next-generation sequencing reads across PODXL aligned to the reference genome in Integrative Genome Viewer (IGV). The antisense strand is indicated as reference. (c) Sanger sequencing confirming the PODXL variant in all affected individuals where DNA was available. The sense strand is indicated. (d) Multisequence alignment showing conservation of the affected amino acid residue, p.442L indicated with an “*”.

Article Snippet: A plasmid containing the full-length human PODXL cDNA (transcript variant 2) was obtained from Origene and was mutated to remove the carboxyl-terminal epitope tags.

Techniques: Sequencing, Variant Assay, Mutagenesis, Next-Generation Sequencing, Residue

Journal: Kidney international

Article Title: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis

doi: 10.1038/ki.2013.354

Figure Lengend Snippet: PODXL rare variants discovered in the FSGS cohort and selected PODXL rare variants from the Exome Sequencing Project (ESP). The number of alleles with the indicated variant found in the 1000 Genomes Project (1KG) and ESP are indicated in columns 7 and 8. At the time of reference, sequence information for 2200 and 10800 haplotypes was available in the 1KG and ESP, respectively. All of these variants were introduced by mutagenesis in plasmids harboring the PODXL gene for further experiments. Amino acid changes are indicated using NCBI accession number NM_005397. DNA coordinates are indicated in reference to human Hg19.

Article Snippet: A plasmid containing the full-length human PODXL cDNA (transcript variant 2) was obtained from Origene and was mutated to remove the carboxyl-terminal epitope tags.

Techniques: Sequencing, Variant Assay, Mutagenesis

Journal: Kidney international

Article Title: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis

doi: 10.1038/ki.2013.354

Figure Lengend Snippet: FSGS-associated genetic variants do not alter the stability of PODXL protein. MDCK cells were co-transfected with GFP and equal amounts of either wildtype PODXL plasmid, PODXL plasmid containing an FSGS-associated variant (p.L442R to p.K515R), or PODXL plasmid containing a control variant from the Exome Sequencing project (p.G112S to p.M490V). After 24 h, the cells were lysed, and the lysates were immunoblotted for PODXL (upper panel) or GFP (lower panel). * indicates amino acid is deleted.

Article Snippet: A plasmid containing the full-length human PODXL cDNA (transcript variant 2) was obtained from Origene and was mutated to remove the carboxyl-terminal epitope tags.

Techniques: Transfection, Plasmid Preparation, Variant Assay, Control, Sequencing

Journal: Kidney international

Article Title: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis

doi: 10.1038/ki.2013.354

Figure Lengend Snippet: The FSGS-associated p.L442R variant induces the formation of PODXL dimers. (a) MDCK cells transfected with either wildtype or p.L442R PODXL plasmid were mock treated or treated with either PNGase F, neuraminidase, or a combination of O-glycosidase and neuraminidase. No differences were observed before or after deglycosylation patterns in both PODXL wildtype and p.L442R mutant on Western blotting. (b) MDCK cells were transfected with either PODXL wildtype plasmid or PODXL p.L442R plasmid. The cells were lysed after 24 h in either mild lysis buffer (TBS + 1% NP-40) or lysis buffer with ionic detergent (RIPA). PODXL was detected using an anti-PODXL antibody. Arrows indicates upper form.

Article Snippet: A plasmid containing the full-length human PODXL cDNA (transcript variant 2) was obtained from Origene and was mutated to remove the carboxyl-terminal epitope tags.

Techniques: Variant Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Lysis

Journal: Kidney international

Article Title: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis

doi: 10.1038/ki.2013.354

Figure Lengend Snippet: The FSGS-associated p.L442R variant does not alter the subcellular localization of PODXL. MDCK cells transfected with PODXL wildtype or p.L442R plasmid. PODXL was labeled with an anti-PODXL antibody (red), F-actin was labeled with phallodin (green), and nuclei were labeled with Hoechst 33342 (blue). (a) Horizontal slices of confocal stacks. (b) Vertical slices of confocal stacks. (c) Transfected MDCK cells were surface biotinylated, and cell surface proteins were pulled down using streptavidin beads. Total PODXL and biotinylated PODXL were visualized by Western blot.

Article Snippet: A plasmid containing the full-length human PODXL cDNA (transcript variant 2) was obtained from Origene and was mutated to remove the carboxyl-terminal epitope tags.

Techniques: Variant Assay, Transfection, Plasmid Preparation, Labeling, Western Blot

Journal: Kidney international

Article Title: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis

doi: 10.1038/ki.2013.354

Figure Lengend Snippet: The FSGS-associated L442R variant does not alter co-localization of PODXL with ezrin at the apical surface. MDCK cells co-transfected with ezrin and either wildtype or p.L442R PODXL plasmid were fixed. PODXL was labeled with an anti-PODXL antibody (red) and GFP-tagged ezrin was labeled with anti-GFP antibody (green). Scale bar: 20 μm.

Article Snippet: A plasmid containing the full-length human PODXL cDNA (transcript variant 2) was obtained from Origene and was mutated to remove the carboxyl-terminal epitope tags.

Techniques: Variant Assay, Transfection, Plasmid Preparation, Labeling

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 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 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