Journal: bioRxiv

Article Title: A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

doi: 10.1101/122580

Figure Lengend Snippet: a. Heatmap representing the 47 phosphatases differentially expressed at transcript and/or protein level at 4h vs. 0h. b. Heatmap showing differential expression of those phosphatases that are upregulated at 4h in the microarray dataset at 4, 8 and 12h relative to 0h. c. Effect of knocking down the 21 phosphatases identified in (b) on clonal growth of keratinocytes. INPP5J was chosen as a control because it did not change in the microarray dataset. Values plotted are average % clonal growth in n=3 independent screens with n=3 independent cultures per screen. Green: SCR control. Red, blue: phosphatases with statistically significant effects on colony formation are shown (red: increase; blue: decrease). Grey: no statistically significant effect. d, e. Effect of knockdowns on clonal growth after 0h, 4h, 8h or 12h in suspension. (d) Representative dishes. (e) Mean % colony formation and individual values (n=3 independent transfections). f, g. RT-qPCR quantification of TP63 ( f ) and TGM1 ( g ) mRNA levels (relative to 18s expression) in the same conditions as in (d) . n =3 independent transfections. h. Epidermal reconstitution assay following knockdown of DUSP6, PTPN1, PPP3CA or PTPN13. n =2 independent transfections. Top row shows representative H&E images. Epidermal thickness was quantified in multiple fields from 8 sections per replicate ± SD relative to scrambled control (SCR). Middle row shows staining for TP63 (pink) with DAPI nuclear counterstain (blue). % DAPI labelled nuclei that were TP63+ was quantified in n =2-3 fields per replicate. Bottom row shows staining for Ki67 (brown) with haematoxylin counterstain (blue). % Ki67+ nuclei was quantified in n=3-6 fields per replicate. Error bars represent mean ± s.d. p -values were calculated using one-way ANOVA with Dunnett’s multiple comparisons test (* p < 0.05; ** p < 0.01; **** p < 0.0001; ns = non-significant).

Article Snippet: Antibodies against the following proteins were used: P-ERK (Cell Signaling # 9101; western blot – 1:1000 dilution), ERK (Cell Signaling # 9102; western blot – 1:1000), P-p38 (Cell Signaling # 9211; western blot – 1:1000), p38 (Cell Signaling # 9212; western blot – 1:1000), α-Tubulin (Sigma # T6199; western blot – 1:5000), MKP-3/DUSP6 (Abcam # ab76310; western blot - 1:1000 and R&D Systems # MAB3576-SP; immunostaining – 1:200), PPTC7 (Abcam # ab122548; western-blot 1:250 and Sigma # HPA039335; immunostaining – 1:200), PTPN1/PTP1B (Sigma # HPA012542; western-blot - 1:500; R&D Systems # AF1366-SP; immunostaining – 1:200), PTPN13 (R&D Systems # AF3577; western-blot – 1:300 and immunostaining – 1:200), PPP3CA/Calcineurin A (Sigma # HPA012778; western-blot 1/1000 and R&D Systems # MAB2839-SP; immunostaining – 1:200), DUSP10 (Abcam # 140123; western-blot - 1:1000 and immunostaining – 1:200), TP63 (SCBT # sc367333; immunostaining – 1:100), Involucrin (SY5, in-house; immunostaining – 1:500) and β1-Integrin (clone P5D2, in-house; immunostaining – 1: 200).

Techniques: Quantitative Proteomics, Microarray, Control, Suspension, Transfection, Quantitative RT-PCR, Expressing, Reconstitution Assay, Knockdown, Staining

Journal: bioRxiv

Article Title: A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

doi: 10.1101/122580

Figure Lengend Snippet: a. Effect of TSA and PKCi on clonogenicity of keratinocytes following suspension in methylcellulose for 12h. b. mRNA levels of IVL, TGM, ITGα6, and TP63 in cells held in suspension for 12h. Cells were treated with TSA, PKCi or DMSO (vehicle control) ( n = 3 independent treated cultures with two technical replicates each). p -values for the comparisons were generated by Tukey’s multiple comparison test. c. Heatmaps showing the effect of knocking down individual phosphatases on mRNA levels of other phosphatases with time in suspension (0h, 4h, 8h, 12h). RT-qPCR is relative to 18s mRNA ( n =3 independent transfections; see Extended Data Table 8 for p -values generated by two-way ANOVA with Dunnett’s multiple comparisons test). d. Western blots showing phosphatase levels in primary keratinocytes upon knockdown of scrambled control (SCR), DUSP6, PPTC7, PTPN1, PTPN13 or PPP3CA and suspension for 0, 4, 8 or 12h. α-tubulin: loading control. e. RT qPCR quantification of phosphatase mRNA levels (relative to 18s mRNA) following doxycycline-induced over-expression of DUSP6, mutant DUSP6 C293S and DUSP10. Cells were treated with 1μg/ml doxycycline for 8h, 24h or 48h ( n =3 independent cultures; see Extended Data Table 9 for p -values generated by two-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: Antibodies against the following proteins were used: P-ERK (Cell Signaling # 9101; western blot – 1:1000 dilution), ERK (Cell Signaling # 9102; western blot – 1:1000), P-p38 (Cell Signaling # 9211; western blot – 1:1000), p38 (Cell Signaling # 9212; western blot – 1:1000), α-Tubulin (Sigma # T6199; western blot – 1:5000), MKP-3/DUSP6 (Abcam # ab76310; western blot - 1:1000 and R&D Systems # MAB3576-SP; immunostaining – 1:200), PPTC7 (Abcam # ab122548; western-blot 1:250 and Sigma # HPA039335; immunostaining – 1:200), PTPN1/PTP1B (Sigma # HPA012542; western-blot - 1:500; R&D Systems # AF1366-SP; immunostaining – 1:200), PTPN13 (R&D Systems # AF3577; western-blot – 1:300 and immunostaining – 1:200), PPP3CA/Calcineurin A (Sigma # HPA012778; western-blot 1/1000 and R&D Systems # MAB2839-SP; immunostaining – 1:200), DUSP10 (Abcam # 140123; western-blot - 1:1000 and immunostaining – 1:200), TP63 (SCBT # sc367333; immunostaining – 1:100), Involucrin (SY5, in-house; immunostaining – 1:500) and β1-Integrin (clone P5D2, in-house; immunostaining – 1: 200).

Techniques: Suspension, Control, Generated, Comparison, Quantitative RT-PCR, Transfection, Western Blot, Knockdown, Over Expression, Mutagenesis

Journal: Scientific Reports

Article Title: Proteins interacting with cloning scars: a source of false positive protein-protein interactions

doi: 10.1038/srep08530

Figure Lengend Snippet: (A), a region within the C-terminus of Flexi cloned TNIP2 is important for its association with PTPN13. Plasmids expressing the six Halo-tagged constructs indicated were transiently transfected in HEK293T cells for Halo affinity purification followed by MudPIT mass spectrometry analysis. Relative amounts of the five prey proteins indicated in enriched using each of these six baits (FDR < 0.05) are indicated according to their relative dNSAF value. Average prey dNSAF values were calculated from between three and six replicate experiments for each bait (see ). Average prey dNSAF values were then normalized to the average bait dNSAF to generate relative dNSAF values. (B), the association of Flexi-cloned TNIP2 and PTPN13 depends on the C-terminal valine cloning scar. Whole cell extracts from HEK293T cells transfected with the indicated constructs were subjected to Halo affinity chromatography and samples were analysed by SDS-PAGE followed by Western blotting. TNIP2 protein was visualized using rabbit anti-TNIP2 or rabbit anti-PTPN13 primary antibodies, and Alexa-680 labeled anti-rabbit secondary antibodies. Note the change in molecular weight of the TNIP2 bait after purification, which involves removal of the 33 kDa Halo tag. Western blots were imaged using a Li-Cor infra-red imaging system. (C), Halo purified proteins from HEK293T cells transfected with Halo-TNIP2 253–429 (5 biological replicates), Halo-TNIP2 253–429 no valine (3 biological replicates) and Halo-PTPN13 (3 biological replicates) were analysed by mass spectrometry. The numbers of distributed MS/MS spectra corresponding to the proteins TNIP2, PTPN13 and STXBP4 from each of these replicates is indicated. None of these proteins were detected in 9 biological replicates of control purifcations using cells expressing the Halo tag alone.

Article Snippet: Rabbit anti-TNIP2 (NBP1-32689) and rabbit anti-PTPN13 (NB100-56139) polyclonal antibodies were from Novus Biologicals.

Techniques: Clone Assay, Expressing, Construct, Transfection, Affinity Purification, Mass Spectrometry, Cloning, Affinity Chromatography, SDS Page, Western Blot, Labeling, Molecular Weight, Purification, Imaging, Tandem Mass Spectroscopy, Control

Journal: Scientific Reports

Article Title: Proteins interacting with cloning scars: a source of false positive protein-protein interactions

doi: 10.1038/srep08530

Figure Lengend Snippet: Part of the PTPN13 ORF coding for a 903 aa region, which included the five PDZ domains, was subcloned into FLAG-pcDNA5/FRT and coexpressed in HEK293T cells with or without Halo-TNIP2 (with the valine cloning scar) as indicated. Lysates were subjected to Halo affinity purification and the resulting eluates analysed by SDS-PAGE and Western blotting. Proteins were detected using anti-FLAG® M2 mouse monoclonal and anti-TNIP2 rabbit polyclonal primary antibodies, and with IRDye® 680LT labeled anti-mouse (red) and IRDye® 800CW anti-rabbit (green) secondary antibodies. Proteins were visualized using a LI-COR® Odyssey® infrared imaging system.

Article Snippet: Rabbit anti-TNIP2 (NBP1-32689) and rabbit anti-PTPN13 (NB100-56139) polyclonal antibodies were from Novus Biologicals.

Techniques: Cloning, Affinity Purification, SDS Page, Western Blot, Labeling, Imaging

Journal: Biomedicines

Article Title: Reversal of Myofibroblast Apoptosis Resistance and Collagen Deposition by Phaseoloidin-Induced Autophagy Attenuates Pulmonary Fibrosis

doi: 10.3390/biomedicines13112679

Figure Lengend Snippet: PTPN13 mediated by phaseoloidin links autophagy and apoptosis. ( A ) Western blot analysis of PTPN13 levels in FasL and different dose phaseoloidin co-treated myofibroblasts ( left ) and quantification of the PTPN13 band intensity ( right ). ( B ) Western blotting reflects the CO-IP assay of PTPN13 binding to p62 under co-treatment with FasL and phaseoloidin or CQ. ( C ) Western blot analysis of PTPN13 levels in FasL and 100 μM phaseoloidin co-treated primary mouse myofibroblasts in the presence or absence of CQ ( top ) and quantification of the PTPN13 band intensity ( bottom ). ( D ) Flow cytometry was used to detect the percentage of apoptosis in the above experimental groups. ( E ) Flow cytometry was used to detect the percentage of apoptosis in groups with or without vector-PTPN13. Data in this figure represent the means ± S.D. (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: A total of 10% of the proteins electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were transferred to PVDF membranes (Bio-Rad) and 5% ( w / v ) skim milk with TBS buffer containing 0.1% ( v / v ) Tween 20 (TBST) TBS buffer for blocking. β-ACTIN monoclonal antibody (1:5000, Proteintech, Wuhan, China), α-SMA monoclonal antibody (1:1000, CST, USA), Caspase3 polyclonal antibody (1:1000, Proteintech, China), collagen I polyclonal antibody (1:1000, Millipore, USA), Bcl-2 polyclonal antibody (1:1000, Proteintech, China), BAX polyclonal antibody (1:1000, Proteintech, China), PTPN13 polyclonal antibody (1:1000, RD, Minneapolis, MN, USA), p70S6K polyclonal antibody (1:1000, CST, USA), elf-4B polyclonal antibody (1:500, ABclonal, Wuhan, China), mTOR monoclonal antibody (1:1000, CST, USA), Pho-mTOR monoclonal antibody (1:1000, CST, USA), AMPK monoclonal antibody (1:1000, CST, USA) and Pho-AMPK monoclonal antibody (1:1000, CST, USA) were incubated overnight at 4 °C.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Flow Cytometry, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MicroRNA‐30e‐5p promotes cell growth by targeting PTPN13 and indicates poor survival and recurrence in lung adenocarcinoma

doi: 10.1111/jcmm.13198

Figure Lengend Snippet: PTPN13 rescued tumour proliferation‐promoting effects by miR‐30e. ( A and B ) The effects of PTPN13 overexpression on cell proliferation in miR‐30e‐transfected A549 and NCI‐H23 cells indicated by MTT assay. ( C and D ) The effects of PTPN13 overexpression on the protein expression of EGFR/AKT pathway in miR‐30e‐transfected A549 and NCI‐H23 cells indicated by western blotting. ( E and F ) The effects of PTPN13 knockdown on the protein expression of EGFR/AKT pathway in sh‐miR‐30e‐transfected A549 and NCI‐H23 cells indicated by Western blotting.

Article Snippet: Lentivirus‐mediated miR‐30e overexpression, miR‐30e shRNA and PTPN13 overexpression vectors, negative control vector (NC) and virion‐packaging elements were from Genechem (Shanghai, China); The primary antibodies of PTPN13 (rabbit monoclonal antibody, ab198882), β‐actin (mouse monoclonal antibody, ab8226), EGFR (rabbit monoclonal antibody, ab52894), AKT(rabbit polyclonal antibody, ab126811), p‐AKT(rabbit polyclonal antibody, ab18206) were from Abcam (Cambridge, MA, USA).

Techniques: Over Expression, Transfection, MTT Assay, Expressing, Western Blot