anti ptpn13  (Proteintech)

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: Cellular and Molecular Life Sciences: CMLS

Article Title: Regulation of Cx36 trafficking through the early secretory pathway by COPII cargo receptors and Grasp55

doi: 10.1007/s00018-024-05440-8

Figure Lengend Snippet: Cx36 and Grasp55 interact via PDZ/PBM binding and colocalize in the Golgi. A GFP directed proximity biotinylation was used to identify proteins regulating the transport of Cx36 in the secretory pathway. Immunostaining confirms correct targeting of V5-TurboID-dGBP and localized biotinylation. Heatmap summarizes the abundance of hits in the BioID screen. Among several secretory pathway proteins we identified Grasp55 in Cx36/Cx36-EGFP transfected conditions. B Streptavidin-affinity-captured proteins were detected on western blots using Streptavidin-HRP, anti-Cx36, anti-V5 and anti-Grasp55. C Immunoprecipitation using GFP trap demonstrates a PDZ dependent binding interaction between Cx36 and Grasp55-GFP. A Grasp55 variant lacking the PDZ binding motif fails to interact with Cx36. A Cx36 mutant lacking the PDZ binding motif does not co-precipitate with full length Grasp55-GFP. D Confocal scan of Cx36 and Grasp55 colocalizing in perinuclear structures. Line scan illustrates the overlap of intensity maxima. E Proximity ligation assay confirms a molecular association of Cx36 and Grasp55 that is diminished when the Cx36/S318ter mutant (lacking PDZ binding motif) is transfected. Difference in PLA labeling was highly significant. F Colocalization of PTPN13 and Cx36 at gap junctions in HEK293T cells. Scale: 10 µm

Article Snippet: Primary antibodies used were Cx36 1:500 (MAB3045; Millipore, Burlington, MA, RRID:AB_94632), Sec22b 1:250 (186,004; Synaptic Systems, Goettingen, Germany), Syntaxin5 1:500 (110 053; Synaptic Systems), Sec24A 1:250 (PA5-66,043; ThermoFisher, RRID:AB_2662296), Sec24B 1:250 (12,042; Cell Signaling Technology, RRID:AB_2797807), Sec24C 1:250 (PA5-59,101; ThermoFisher, RRID:AB_2647077), Golgin97 1:250 (PA5-84,015; ThermoFisher, RRID:AB_2791167), GM130 1:250 (12,480; Cell Signaling Technology, RRID:AB_2797933), PTPN13 1:250 (PA5-72,906; Thermofisher, AB_2718760), Grasp55 1:250 (MA5-24,642; ThermoFisher, RRID:AB_2637257) and Grasp55 1:500 (10,598–1-AP; Proteintech, RRID:AB_2113473).

Techniques: Binding Assay, Immunostaining, Transfection, Western Blot, Immunoprecipitation, Variant Assay, Mutagenesis, Proximity Ligation Assay, Labeling