Denaturing buffer

Solubilized proteins from HEK293T cells transfected with expression plasmids for WT or mutant Slitrks, were first denatured by adding 10x denaturing buffer (New England Biolabs) and heating to 100°C for 10 min. For endoglycosidase H (Endo H) treatment, denatured protein was treated with 1 μl of enzyme and incubated at 37°C for 1 h. For PNGase F treatment, denatured protein was incubated with 1 μl of enzyme in the presence of 2 μl of NP-40 (100%) at 37°C for 1 h. Thereafter, enzyme-treated proteins, together with an equal amount of untreated proteins, were analyzed by immunoblotting with the indicated antibodies followed by enhanced chemiluminescence (ECL) detection.

First, we performed de-N-glycosylation of human plasma using PNGase F enzyme according to manufacturer’s instruction. Briefly, 2.5 μL of plasma samples and commercially available human plasma-derived purified proteins were denatured with 3 μL of 10X Denaturing buffer (New England BioLabs) and 24.5 μL of distilled water (DW) was added to make a 30 μL total reaction volume. Samples were incubated at 99°C for 10 min in a heating block, and then cooled down at room temperature (RT) for 10 min. Supernatants were transferred to Amicon Ultra-10 kDa MWCO (Millipore). 6 μL of 10X GlycoBuffer 2 (New England BioLabs) and 24 μL of DW were added to MWCO to make a total reaction volume of 60 μL. 2 μL of PNGase F was added and incubated at 37°C for 4 h. After deglycosylation, released N-glycan mixture was collected by centrifugation at 14,000×g for 15 min. To fully remove denaturation buffer, 400 μL of 100 mM HEPES buffer (pH 7.8) was added and washed by centrifugation at 14,000×g for 15 min at least three times.

HeLa C1 cells35 (link) were transfected on days 1 and 2 post-plating with 50 nM of RISC Free control siRNA (D-001220-01), CLEC16a siRNA (D-022485-18) or a pool of siRNA directed against RAB1A and B (L-008283-00, L-008958-01) (Dharmacon/GE Healthcare) using Oligofectamine (day 1) (Life technologies) and Lipofectamine 2000 (day 2) (Life technologies) and re-plated into 6-well cluster plates (day 3). Following treatment with 1 μM D/D solubiliser (FKBP AP21998) (Clontech), the amount of the eGFP tagged reporter construct remaining at each time point was measured by flow cytometry on a LSR II (Beckton Dickinson) and expressed as a percentage of the zero time point for each. For analysis of LAMP-1 glycosylation levels, B6.Clec16a^GT/GT MEFs and HeLa-CLEC16A cells were lysed in 1% SDS, 10× Denaturing buffer (New England Biolabs) and lysates were incubated with Endoglycosidase H and Peptide-N Glycanase at 37 °C for 5 hours. Lysates were probed for LAMP-1 protein levels following resolution by SDS-PAGE.