Anti gpc1 antibody

Total proteins samples were extracted from primary tissues or from cell lines using lysis buffer containing phenylmethyl sulfonyfluoride (PMSF). The samples were mixed with loading buffer and denatured, separated by electrophoresis in a 10% SDS-PAGE gel, and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 2 h, incubated with anti-GPC1 antibody (Abcam, Cambridge, UK) or anti-GAPDH antibody (Abcam, Cambridge, UK) overnight at 4 °C. Signals were revealed after incubation with anti-rabbit IgG secondary antibody coupled to peroxidase by using ECL.

LP (GEM) was firstly prepared. In brief, 120 mg of hydrogenated soybean phospholipid (HSPC) and 34 mg of cholesterol were dissolved in 6 mL of chloroform, and 2 mL of GEM hydrochloride solution (3 mg/mL) was added into the above lipid solution. Subsequently, a uniform emulsion was formed by the ultrasonic shaking for 10 min, and then chloroform was removed by reduced pressure distillation at 45°C for 1h. After the temperature was raised to 60°C, 2 mL of isothermal PBS solution was added to hydrate for 1 h, followed by ultrasonication for 3 min, and repeated freeze-thaw for 4 cycles. Lastly, LP (GEM) was were obtained by incubating with 12 mg of carboxylated distearoyl phospho-ethanolamine-polyethylene glycol (DSPE-PEG2000) for 15 min in a 60°C water bath. Similarly, unloaded LPs were prepared as described above but without the addition of GEM.
Afterwards, 900 μL of LP (GEM) solution was incubated with 100 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mg/mL) and 50 μL of N-Hydroxysuccinimide (10 mg/mL) by shaking at 400 rpm in a constant temperature shaker at 25° C for 3 h, then, 10 μL of anti-GPC1 antibody (10 nmol, Abcam, Cambridge, MA, USA) was added dropwise into the above mixture. After shaking for 2 h, the mixture was blocked with bovine serum albumin (BSA), and GPC1-LP (GEM) was obtained by shaking for 10 h.