Hrp conjugated anti rabbit igg

Caco-2 cells were treated with 5 mM β-xyloside or 10 µM Stattic for 4 h at 37 ℃, and then were incubated with GST-rTsCTL (5 µg/mL) at 37 ℃ for 2 h [17 (link)]. The Caco-2 cells were lysed in RIPA buffer, ultrasonicated in an ice bath for 30 s and centrifuged at 12 000 × g for 15 min to remove any cell fragments. The cell soluble proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA) in the wet transfer cell (Bio-Rad, USA). The membrane was blocked with 5% skim milk in TBST at 37 °C for 2 h and incised into strips. The strips were probed with antibodies against syndecan-1 (1:1000), total STAT3 (t-STAT3; 1:1000), phosphorylated STAT3 (p-STAT3; 1:5000, Abmart, China), occludin (1:500), claudin-1 (1:200), claudin-2 (1:200) (ThermoFisher, USA), and anti-β-Actin antibody (1:1000) overnight at 4 °C [6 (link)]. After washes with TBST three times, the strips were incubated at 37 °C for 1 h with HRP-conjugated anti-rabbit IgG (1:10 000; Southern Biotech). And then, Omni-ECL reagents (Epizyme, Shanghai, China) were used to visualize the reactive bands, and the relative intensities of each band were analyzed using the Image J software (National Institutes of Health, USA) [36 (link)].

Samples of treated cells, exosomes and MVs fraction were lysed in RIPA buffer(Santa Cruz, USA) for 20 min on ice and cleared lysate was collected by centrifugation for protein separation on 10% SDS-PAGE gels(Bio-Rad, USA). The proteins were transferred onto Immobilon-FL PVDF Transfer Membrane(Millipore) and detected with appropriate primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Southern Biotech) secondary antibodies. Membranes were exposed using an enhanced chemoluminescence(NcmECL Ultra) system(NCM Biotech, Suzhou, China) and band intensities were quantified by ImageJ software(NIH). Primary antibodies against CD63(BD Biosciences), CD9(Sigma, #SAB4503606), DAF(#31759), CVB3 VP1(Dako,#M706401-2), Annexin A1(#32934), Annexin A5(#8555), GM130(#12480), Calnexin(#2679) (all from Cell Signaling Technology, USA), CAR(#ab272711), Alix(#ab186429), SCARB2(#ab240186), Tyro3(#ab109231), GAPDH(#ab8245)(all from Abcam, USA), CAV16 VP1(GTX132338), CAV16 3AB(GTX132344), Zika Envelope(GTX133314), Zika Capsid(GTX133317)(All from GeneTex, Inc. Taiwan, China).

Epichaperome abundance in CD34 + blasts in WCM254 at baseline and on day 5 after starting treatment was measured by using a capillary-based platform that combines isoelectric focusing (IEF) with immunoblotting capabilities as described before^{1 (link)}. Cells were lysed in 20 mM HEPES pH 7.5, 50 mM KCL, 5 mM MgCl₂, 0.01% NP40, 20 mM Na₂MoO₄ buffer containing protease and phosphatase inhibitors. Total protein assay to detect epichaperome was performed on an automated system, NanoPro 1000 Simple Western (ProteinSimple), for charge-based separation. Samples were loaded into capillaries (ProteinSimple) and separated based on their isoelectric points. Immobilization was performed by UV-light embedded, followed by labeling with anti-HSP90β (SMC-107A, StressMarq Biosciences) and subsequently with HRP-conjugated anti-Mouse IgG (1030-5, SouthernBiotech) or with HRP-conjugated anti-Rabbit IgG (4010-05, SouthernBiotech). Protein signals were quantified by chemiluminescence using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific), and digital imaging and associated software (Compass) in the Simple Western system, resulting in a gel-like representation of the chromatogram.