Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Confocal microscopy analysis of the largest extracellular domain of NaPi2b in ovarian carcinomas cells OVCAR-4 that were either alive (A–D) or fixed and permeabilized (E–H) and imaged in the Z-stacking mode. (A,E) Blue channel (Hoechst); (B,F) Red channel (PI); and (C,G) Green channel (Alexa Fluor 488), ECD-NaPi2b–mAbs L2 (20/3) directed to ECD of NaPi2b; (D,H) Merged images; On the top ( Y -axis and Z -axis combined) and right ( X -axis and Z -axis combined) of each picture, a composite image comprising of numerous photographs obtained at various focus distances is shown. White arrows indicate the position of NaPi2b on the cell membrane.

Article Snippet: Site-directed anti-C-NaPi2b/SLC34A2 mAbs (D3V3I, cell signaling) detect the C-terminal domain of the NaPi2b phosphate transporter only in cells that are fixed and permeabilized , but not in live PI-negative OVCAR-4 cells ( ).

Techniques: Confocal Microscopy

Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Confocal microscopy analysis of the N-terminal domain of NaPi2b in ovarian carcinomas cells OVCAR-4 that were either intact (A–D) or fixed and permeabilized (E–H) and imaged in the Z-stacking mode. (A,E) Blue channel (Hoechst); (B,F) Red channel (PI); and (C,G) Green channel (Alexa Fluor 488), N-NaPi2b–mAbs N-NaPi2b (15/1) directed to the N-terminal domain of NaPi2b; (D,H) Merged images; On the top ( Y -axis and Z -axis combined) and right ( X -axis and Z -axis combined) of each picture, a composite image comprising of numerous photographs obtained at various focus distances is shown. White arrows indicate the position of NaPi2b on the cell membrane.

Article Snippet: Site-directed anti-C-NaPi2b/SLC34A2 mAbs (D3V3I, cell signaling) detect the C-terminal domain of the NaPi2b phosphate transporter only in cells that are fixed and permeabilized , but not in live PI-negative OVCAR-4 cells ( ).

Techniques: Confocal Microscopy

Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Confocal microscopy analysis of the C-terminal domain of NaPi2b in ovarian carcinomas cells OVCAR-4 that were either alive (A–D) or fixed and permeabilized (E–H) and imaged in the Z-stacking mode. (A,E) Blue channel (Hoechst); (B,F) Red channel (PI); and (C,G) Green channel (Alexa Fluor 488), C-NaPi2b–mAbs D3V3I (Cell Signaling, United States) directed to the C-terminal domain of NaPi2b; (D,H) Merged Images; On the top ( Y -axis and Z -axis combined) and right ( X -axis and Z -axis combined) of each image, a composite image comprising of numerous photographs obtained at various focus distances is shown. White arrows indicate the position of NaPi2b on the cell membrane.

Article Snippet: Site-directed anti-C-NaPi2b/SLC34A2 mAbs (D3V3I, cell signaling) detect the C-terminal domain of the NaPi2b phosphate transporter only in cells that are fixed and permeabilized , but not in live PI-negative OVCAR-4 cells ( ).

Techniques: Confocal Microscopy

Journal: Frontiers in Molecular Biosciences

Article Title: Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells

doi: 10.3389/fmolb.2022.895911

Figure Lengend Snippet: Topological model of NaPi2b with color-coded antigenic regions recognized by available monoclonal analytical and therapeutic antibodies targeting different extramembrane domains. The epitope’s region in ECD of NaPi2b for mAbs, generated by: , 188–300 aa, marked in light purple; , 320–361 aa, marked in green; , 323–330 aa, marked in purple; , , Kiyamova et al. (2008), , , , , 324–338 aa, marked in red; Epitope region in N-termini and C-termini of NaPi2b for mAbs, generated by: Gryshkova et al. (2011), Cell Signaling, Abcam, and others against N-terminal domain of NaPi2b, 1–100 aa, marked in orange; Cell Signaling, Abcam, and others against С-terminal domain of NaPi2b, the region is unknown, marked in lavender. Positions of cysteine and asparagine amino acid residues potentially contributing to the oxidative folding and formation of disulfide bonds and N-linked glycosylation of the largest ECD, and palmytoilation site in C-terminal domain are also shown.

Article Snippet: Site-directed anti-C-NaPi2b/SLC34A2 mAbs (D3V3I, cell signaling) detect the C-terminal domain of the NaPi2b phosphate transporter only in cells that are fixed and permeabilized , but not in live PI-negative OVCAR-4 cells ( ).

Techniques: Generated

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Intersection genes expression and Kaplan-Meier survival analysis based on TCGA clinical cohorts. A was the Venn plot of overlapped DEGs of GSE3678, GSE29265, GSE50901, and GSE33630; B-D were box plot of expression of SLC34A2, SLC4A4, and SLC25A15 in tumor and matched normal thyroid; E-G were the survival curves of SLC34A2, SLC4A4, and SLC25A15, respectively. Black box symbolized the tumor, grey box symbolized the normal. Red line symbolized high expression, blue line symbolized low expression. TCGA: The Cancer Genome Atlas; DEGs: differentially expressed genes.

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Expression of SLC34A2 (A and D), SLC4A4 (B and E), and SLC25A15 (C and F) in tumor tissues and adjacent normal thyroid tissues. A-C were the mRNA expression analysis by RT-PCR (N=8); D-F were the representative protein bands of western blotting. “*” represented P < 0.05.

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Association between the expression of SLC34A2 with clinicopathological characteristics of PTC patients (mean ± SD, n, %)

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Logistic regression analysis for predictors of the capsular invasion risk of PTC patients

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques:

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Logistic regression analysis for predictors of the extra-thyroid metastasis risk of PTC patients

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques:

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Logistic regression analysis of high UIC for high SLC34A2 expression risk of PTC patients

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Representative immunohistochemical staining for SLC34A2 and SLC4A4 in tumor tissues and adjacent normal thyroid tissues of PTCs who suffered with capsule invasion and extra-thyroid metastasis, respectively. Scale is 400× for each picture. PTC, papillary thyroid carcinoma.

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques: Immunohistochemical staining, Staining

Journal: Journal of Cancer

Article Title: SLC34A2 Up-regulation And SLC4A4 Down-regulation Correlates With Invasion, Metastasis, And The MAPK Signaling Pathway In Papillary Thyroid Carcinomas

doi: 10.7150/jca.56730

Figure Lengend Snippet: Correlation of SLC34A2 and SLC4A4 expression with MAPK signaling pathway of PTCs. A and B namely the correlation between p-ERK with SLC34A2 (r=0.225, P=0.420) and SLC4A4 (r=-0.354, P=0.215); C and D namely the correlation between p-JNK with SLC34A2 (r=0.437, P=0.118) and SLC4A4 (r=-0.696, P=0.004); E and F namely the correlation between p-P38 with SLC34A2 (r=0.336, P= 0.240) and SLC4A4 (r=-0. 534, P=0.049). PTC, papillary thyroid carcinoma.

Article Snippet: The membranes were blocked with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with primary antibodies against SLC34A2 (dilution 1:1000; D6W2G, CST, US), SLC4A4 (dilution 1:1000; ab187511, Abcam, UK), SLC25A5 (dilution 1:1000; ab228604, Abcam, UK), ERK (dilution 1:500; bsm-33337M, Bioss, China), p-ERK (dilution 1:500; bs-1522R, Bioss, China), JNK (dilution 1:500; E-AB-60070, Elabscience, China), p-JNK (dilution 1:500; BM4380, Boster, China), P38 (dilution 1:500; E-AB-32459, Elabscience, China), p-P38 (dilution 1:500; E-AB-21027, Elabscience, China), β-actin (dilution 1:2000; 17AV0412, Zhongshan Golden Bridge, China).

Techniques: Expressing

Journal: Scientific Reports

Article Title: New insights into the resistance mechanism for the BceAB-type transporter Sa NsrFP

doi: 10.1038/s41598-022-08095-2

Figure Lengend Snippet: ( a ) Fold of resistance of L. lactis NZ9000NsrFP and NZ9000NsrF H202A P (hatched bars) against L. lactis NZ9000Cm calculated with the determined IC 50 of ramoplanin A2 (yellow), vancomycin (orange), lysobactin (green), bacitracin (blue) and bacitracin with ZnCl 2 (dark red). Values for nisin and gallidermin were taken from Reiners et al. 34 and marked with an asterisk. Values were calculated from at least 4 independent measurements and are also listed in Table . A two-sided Students t-test was performed with the IC 50 data obtained for Sa NsrFP and Sa NsrF H202A P. Significance was marked with an asterisk. p-values were listed in a separate table (SI Table ) in the supplement. ( b ) Expression of Sa NsrFP (1) and Sa NsrF H202A P (2) and the empty vector pIL-SV (3) in L. lactis NZ9000, monitored via western blot with a polyclonal antibody against the extracellular domain of Sa NsrP. Loaded are purified membranes from the corresponding strains. A nonlinear regression curve fit and a two-sided, unpaired Students t-test was performed using Graphpad Prism version 9.2.0 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com .

Article Snippet: A polyclonal antibody against the extracellular domain of Sa NsrP was used to detect the expressed Sa NsrFP protein (Davids Biotechnologie, Regensburg, Germany).

Techniques: Expressing, Plasmid Preparation, Western Blot, Purification, Software