Lamp2

Pancreatic tissue were isolated from animals, manually cut into 10mm cubes, incubated in 2% paraformaldehyde in PBS for 30 minutes on ice, and place din 15% sucrose in PBS overnight at 4°C. Tissue pieces were then snap frozen on dry ice in OCT Compound in cryomolds (Sakura Finetek, Torrance, CA), and placed on slides after being cut into thin sections at −20°C. Tissue sections were permeabilized using TBS and 0.05% Saponin (TBS-S) and quenched with TBS-S with 50mM NH4Cl and 5% goat serum. Tissues were incubated with primary antibody (Amylase: 1:200 (Sigma Aldrich), Trypsin 1:1000 (previously made in laboratory), Lamp-2: 1:100 (Sigma Aldrich) diluted in TBS-S overnight at 4°C and incubated with secondary antibody with Alexa Fluor 488 at 1:500 in TBS-S. Slides were mounted using Vectashield hard set mounting medium with DAPI (Vector Labs, Burlingame, CA), examined using fluorescence microscope (Zeiss Axiophot, Oberkochen, Germany), and imaged using images digital camera (Spot Imaging, Sterling Heights, MI).

Pancreatic tissue was fixed in 4% paraformaldehyde, and after overnight fixation, tissues were embedded in paraffin. The tissue slides were de-paraffinized in xylol for 30 min and rehydrated in decreasing concentrations of ethanol before they were transferred to 1x phosphate buffer saline (PBS). Antigen was retrieved by cooking in 10 mM citrate buffer in a pressure cooker for 5 min. Tissue was blocked overnight in 1x aurion BSA. Tissue slides were incubated with primary antibodies a combination of trypsin (AB 1823, Chemicon, CA, USA) with GFP (Cat no # MAB3580, Millipore), and LAMP2 (cat no # L0668, Sigma-aldrich, St. Louis, Germany) with GFP for 2 h followed by corresponding secondary antibodies Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunReasearch, Suffolk, UK) and FITC-conjugated goat anti-mouse IgG (Jackson ImmunReasearch, Suffolk, UK) for 1 h, respectively. Primary antibodies were used at a dilution of 1:100, and secondary antibodies were used at a dilution of 1:200. DAPI is used to stain nuclei. Tissues were mounted with mowiol embedding media. Images were taken by Leica SP5 at 400× magnification. Co-localization was analysed with ImageJ using JACoP plug-in. The percentage of trypsin or LAMP2 positive dots were plotted against LC3 positive dots and graphically shown as co-localized [33 (link)].

Antibodies used for immunoblot are as follows: Egr1 (Cell Signaling 4154, 1:1000); cMyc (Cell Signaling 5605, 1:1000); Rb1cc1 (Cell Signaling 12436, 1:1000); Wnt10a (Santa Cruz sc-376028, 1:500); Ulk1 (Cell Signaling 8054T, 1:1000); Akt1 (Cell Signaling 2938, 1:1000); CD28 (Cell Signaling 38774S, 1:1000); Lamp2 (Sigma L0668, 1:1000); Cathepsin C (Santa Cruz 74590, 1:500); Clathrin (Cell Signaling 2410, 1:1000).

Doxorubicin was purchased from LC laboratories. Other reagents were purchased as follows: sunitinib (Sigma Aldrich), paclitaxel (Sigma Aldrich), Torin 1 (Tocris Bioscience), bafilomycin A1 (LC laboratory), E64d, pepstatin (Sigma Aldrich), concanamycin A (Santa Cruz). Lysotracker Red (DND-99), lysosensor DND-189, acridine orange, Dextran, and Oregon Green 514 were purchased from Invitrogen. Antibodies: LC3 (previously developed^{22 (link)}), p62 (Abnova), GAPDH (Fitzgerald Industries), pS6 (240/244), S6, pS6K (T389), S6K, p4EBP1, 4EBP1 (Cell Signaling), LAMP-1 (Hybridoma bank), LAMP-2 (Sigma Aldrich), ATP6V0D1, ATP6V0A2, ATPV1D, ATPV1B2 (Abcam).