Kpl detector block solution

Organoids were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, 78501) supplemented with protease inhibitors (Roche, 05 892 970 001) according to the manufacturer's protocol. Cell lysates were resuspended in 40 μl Laemmli Loading Buffer containing β-mercaptoethanol (Bio-Rad Laboratories, 1610730) before western blot analysis. Samples were loaded onto 4–20% Tris-Glycine Gradient Gels (Invitrogen) and run at 80 volts for approximately 3 hours and transferred to nitrocellulose membranes (Whatman Protran, 0.45 μM) at 105 volts for 1.5 hours at 4°C. Membranes were blocked for 1 hour at room temperature using KPL Detector Block Solution (Kirkegaard & Perry Laboratories, Inc., 718300). Membranes were incubated for 16 hours at 4°C with either 1:100 dilution of rat anti-PD-L1 (Novus Biologicals, NBP1-76769), or 1:2000 dilution of mouse anti-GAPDH (Millipore, MAB374) antibodies followed by 1 hour incubation with a 1:1000 dilution anti-mouse, or -rat Alexa Fluor 680 (Invitrogen). Blots were imaged using a scanning densitometer along with analysis software (Odyssey Infrared Imaging Software System) and the ratio of PD-L1/GAPDH was calculated using ImageJ.

Organoids and monolayers were harvested in cold DMEM/F12 and lysed in M-PER Mammalian Protein Extraction Reagent (Thermofisher) supplemented with protease inhibitors (Roche) according to the manufacturer’s protocol. Cell lysates were suspended in 40 μL of Laemmli Loading Buffer containing β-mercaptoethanol (BioRad). Samples containing 20 μg of protein were then loaded onto 1 4–20% Tris-Glycine Gradient Gels (Invitrogen) and run at 120 V for 1.5 hours before transferring the protein onto nitrocellulose membranes (Whatman Protran, 0.45 μM) at 105 V for 1.5 hours at 4°C. Membranes were blocked for 1 hour at 23°C using KPL Detector Block Solution (Kirkegaard & Perry Laboratories, Inc.). Next membranes were incubated overnight at 4°C with a 1:1000 dilution of anti-PD-L1 (Novus, NBP1-76769) a 1:1000 dilution of anti-Shh (Novus, AF464) or 1:2000 dilution of anti-GAPDH (Millipore, MAB374). The membranes were washed 3 times for 5 minutes each. Following this, the membranes were incubated with a 1:1000 dilution anti-mouse, 1:1000 dilution anti-goat or anti-rabbit Alexa Fluor 680 (Invitrogen). The blots were then imaged using a scanning densitometer along with analysis software (Odyssey Infrared Imaging Software System).

Cell lysates were mixed (1:1 v/v) with Laemmli loading buffer plus β-mercaptoethanol (Bio-Rad Laboratories, CA) before western blots were performed. Samples were then loaded into 4–20% Tris–glycine gradient gels (Invitrogen) and ran at 80 V for 2.5 h at room temperature and transferred to nitrocellulose membranes (Whatman Protran, 0.45 µM) at 105 V for 1 h 15 min at 4 °C. Membranes were then blocked at room temperature using KPL Detector Block Solution (Kirkegaard & Perry Laboratories, Inc.) for 1 h and subsequently incubated at 4 °C for 16 h with a 1:1000 dilution of anti-GAPDH (Thermo Fisher Scientific), 1:1000 anti-AKT (Cell Signaling), 1:1000 phospho AKT (Cell Signaling), followed by 1 h incubation with a 1:1000 dilution anti-mouse, anti-rabbit, or anti-rat Alexa Fluor 680 (Invitrogen) at room temperature. Blots were imaged using a scanning densitometer along with analysis software (Odyssey Infrared Imaging Software System) and the ratio of individual antibody/GAPDH was calculated using ImageJ.