Hrp conjugated goat anti rabbit igg

mRNA was purified using PolyATtract® mRNA Isolation Systems (Promega, Z5310) following the manufacturer’s instructions. mRNA was denatured at 95 °C for 5 min and loaded onto a Hybond-N+ membrane (GE HealthCare, RPN303B). After closslinking under ultraviolet radiation, the membrane was blocked with 5% non-fat milk (Bio-Rad) for 1 h, incubated with rabbit anti-m6A polyclone antibody (Synaptic Systems, 202003) at 4 °C overnight. Then the membrane was incubated with HRP-Conjugated goat anti-rabbit IgG (CWbio, CW0156) at room temperature for 2 h. After being incubated with a Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500), the immunocomplex was photographed using the ECL imaging system. Finally, the membrane was stained with 0.02% methylene blue to eliminate the difference in mRNA amount.

For the pharmacokinetic studies, purified Ex-DARP-FGF21 with a His-tag was administered subcutaneously to the C57BL/6 mice at a dose of 10 nmol/kg. Approximately 40 μL of blood was collected from the orbital venous plexus at different time points (1, 3, 8, 12, 24, 36, 48, and 72 h). The plasma was separated by centrifugation at 4,000 g for 10 min at 4°C and then diluted with PBS. The plasma concentration of the fusion protein was measured following the method described by Schellenberger et al. (33 (link)). Briefly, an anti-exendin-4 mouse monoclonal antibody (Abcam, UK) was diluted with a carbonate-bicarbonate buffer (pH 9.6), immobilized in a 96-well plate, and incubated overnight at 4°C. It was then blocked with a reagent (PBS with 10% chicken serum). After washing three times, standard and plasma samples were added to the wells and incubated at 25°C for 2 h. The bound fusion proteins were detected using a rabbit anti-FGF21 antibody (Abcam, UK) and HRP-conjugated goat anti-rabbit IgG (CWBIO, China). Subsequent procedures were performed as previously described. The concentration of the fusion protein was calculated based on a standard curve. The half-life of the fusion protein was determined using the PKSolver program in Microsoft Excel.

The 4G10 antibody for phosphotyrosine was purchased from Millipore (Boston, MA). The antibodies for paxillin, p130Cas, and p130Cas Y249 were purchased from BD Biosciences (Franklin Lakes, NJ). The antibodies for paxillin Y31, FAK Y397, and FAK Y576/577 were purchased from Abcam (Cambridge, UK). The antibodies for paxillin Y118 was purchased from Cell Signing Technology (Boston, MA) and FAK from Proteintech (Rosemont, IL). Alexa Flour 546 phalloidin and Alexa Fluor 488 goat anti-mouse IgG were purchased from Life Technologies (New York, USA). HRP Conjugated goat anti-rabbit IgG, and HRP Conjugated goat anti-mouse IgG were purchased from CW Biotech (Beijing, China).
Human umbilical vein endothelial cell line ECV-304 was maintained in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO₂.

Equal amounts of extracted intracellular proteins were analyzed on 15% (w/v) SDS polyacrylamide gel electrophoresis (PAGE), after electrophoresis, gels were stained with Coomassie blue R-250 for 2 h. Then 0.25 M KCl decolorizing solution was added and decolorized for many times until the protein band was clearly visible. For Western blot assay, the proteins were transferred to PVDF membrane and the membrane was incubated at 37 °C with 50 mL 5% (w/v) skimmed milk for 2 h. And then the membrane was washed 3 times with PBS for 15 min each time. Afterward, the membrane was incubated with a 1:3000 dilution of pGH polyclonal antibody [33 ] overnight at 4 °C and incubated with HRP conjugated goat anti-rabbit IgG (CWBIO, China) at a dilution of 1:5000 at 37 °C for 1 h. After each incubation, the membrane was washed 3 times with PBST for 15 min each time. Immunoreactive bands were visualized with Enhanced HRPDAB Chromogenic Substrate Kit (TIANGEN, China). Protein bands on SDS–PAGE were estimated by ImageJ software.