Xcell iitm blot module system

Forty-eight hours after transfection, medium was collected and centrifuged at 13,800× g for 5 min at 4 °C. Cells were washed in phosphate-buffered saline buffer (PBS) and lysed in ice-cold RIPA lysis buffer, supplemented with cOmplete Mini protease inhibitor cocktail tablets. The cells were then incubated 30 min on ice and centrifuged at 13800 × g for 15 min at 4 °C. The lysate (detergent-soluble) fraction was removed and the pellet washed twice with PBS. To all fractions were added LDS sample buffer and NuPAGE reducing agent (Invitrogen), heated to 56 °C for 15 min (5 min of boiling at 96 °C for the pellet fraction), separated by SDS-PAGE (NuPAGE, 4–12% Bis-Tris gels), and transferred to PVDF membranes using XCell IITM Blot Module system (Invitrogen) according to the manufacturer’s manual. Specific antibodies and enhanced chemiluminescence were used to detect and visualize the proteins of interest. Blots were visualized using a LAS-1000 imager and Image Reader LAS-1000 Pro v2.6 software (Fujifilm, Tokyo, Japan), and quantification of protein bands was performed using Multi Gauge v3.0 software (Fujifilm).

Immortalized mouse mesenteric lymphatic endothelial cells from confluent 9.6 cm² wells (six-well plates) were lysed using RIPA buffer (Thermo Fisher Scientific, 89900) plus 1X Halt protease and phosphatase inhibitors with EDTA (Thermo Fisher Scientific) and cleared by centrifugation. Cell lysates were subjected to Western blot analysis using the XCell II^TM Blot Module system (Invitrogen, EI9051). PVDF membranes were probed with primary antibodies against PROX-1 and β-actin as a loading control followed by Licor IRDye secondary antibodies. Membranes were imaged using a LI-COR Odyssey CLx imager with near-infrared Western blot detection and analyzed using ImageStudioLite software.