Pmsf protease inhibitor

Transduced cells were lysed in RIPA buffer containing PMSF protease inhibitor (Roche, Mannheim, Germany) and resolved on a 4%–12% polyacrylamide sodium dodecyl sulfate gel. Western blotting and enhanced chemiluminescence (ECL) detection system (Pars Toos biotechnology Co., Mashhad, Iran) were used with HRP-conjugated anti-His tag antibody to detect N-terminal His tag on chimeric LAMP2b-DARPin protein.

Immunoblotting was performed according to a standard protocol. Briefly, the cells were lysed with a RIPA lysis buffer (150 mM NaCl, 0.5% deoxycholic acid, 50 mM Tris-HCl [pH 7.5], 1% NP-40, and 0.1% sodium dodecyl sulphate [SDS]) containing a protease inhibitor mixture comprising 1 mM Na₃VO₄, 10 mM NaF, and 1 mM PMSF protease inhibitor (Boehringer Mannheim, Indianapolis, IN, USA). Cell lysates that were boiled in sample buffer were run through SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes. Nonspecific reactions of the membranes were blocked with 5% skim milk, followed by incubation with primary antibodies overnight at 4°C. Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NF-κB p65, p-NF-κB p65, and E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). The incubated membranes were reacted with secondary antibodies in 5% skim milk at room temperature for 1 hour. Horseradish peroxidase (HRP)-linked immunoglobulin G (IgG) for various animals was purchased from Cell Signaling Technology. The blots were developed using an enhanced chemiluminescent HRP substrate (Thermo-Fisher Scientific/Invitrogen, Waltham, MA, USA).

Immunoblotting was performed according to the standard protocol. Briefly, the cells were lysed with a lysis buffer (150 mM NaCl, 1% deoxycholate, 20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1% Triton X-100, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate) containing a protease inhibitor mixture comprising 1 mM Na₃VO₄, 10 mM NaF, and 1 mM PMSF protease inhibitor (Boehringer Mannheim, Indianapolis, IL, USA), 1 µg/mL of leupeptin, and 1 µg/mL of aprotinin phosphatase inhibitors (Calbiochem, La Jolla, CA, USA). Cell lysates boiled in sample buffer were size-separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4°C. Then, the membranes were incubated with secondary antibodies in 5% skim milk for 1 hour at room temperature. The blots were developed using an enhanced chemiluminescent HRP substrate (Invitrogen). Images of the immunoblotting were digitized to compare the strength of the expression levels of targeted molecules using Image J (National Institutes of Health [NIH], Bethesda, MD, USA). The quantified relative strengths of the expression levels of the target molecules were obtained from the immunoblotting images using the Gels menu in Image J.