Ha 11mms 101r

Western blot method was done accordingly to the previously published protocol [28 ]. In brief, cells were washed with PBS and subsequently lysed in the sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.002% bromphenol blue and 0.06 M Tris HCl, pH 6.8), sonicated and thermally denatured. Electrophoretically separated proteins, in the SDS-PAGE gels, were transferred onto Immobilon-P PVDF Membrane (IPVH00010, Millipore) and detected by primary and corresponding HRP-conjugated secondary antibodies on Fusion SL imaging system (Vibler), using Immobilon Western Chemiluminescent HRP Substrate (Merck, WBKLS0500). Western blot signals were quantified using ImageJ software. Antibodies are listed in the Table 2. Western blot membranes are presented together with molecular mass markers in the Additional file 1.
Table 2

Antibodies

Antibody	Manufacturer	Dilution and application	Reference
LRP6 (C47E12)	cs-3395, Cell Signaling Technology	1:1000, WB	[28 ]
LRP6 (Phospho-S1490)	cs-2568, Cell Signaling Technology	1:1000, WB	[28 ]
β-actin	CS-4970, Cell Signaling Technology	1:3000, WB	[26 (link)]
DVL-2	CS-3216, Cell Signaling Technology	1:1000, WB	[26 (link)]
DVL-3	CS-3218, Cell Signaling Technology	1:1000, WB	[26 (link)]
HA-11	MMS-101R, Covance	1:2000 WB; 1:500, IF	[28 ]
a-mouse IgG HRP	A4416	1:4000 WB	–
a-rabbit IgG HRP	A0545	1:4000 WB	–
a-mouse Alexa Fluor™568	A10037, Thermo Fisher Scientific	1:600 IF	–

Lysates of the patients P1 and P2 and control fibroblasts as well as HeLaM cells were used for immunoblot analysis with anti-human VPS11 (WH0055823M1; Sigma-Aldrich), anti-human VPS41 (sc-377 118; Santa Cruz) or antibodies to VPS33A, VPS 18, HA and actin as described previously (10 ). In addition, lysates from the transfected HeLaM cells were subjected to immunoblot analysis with anti-HA (HA.11, MMS-101R; Covance) and anti-actin (A2066; Sigma-Aldrich) antibodies. In experiments to examine the effects of the proteasome inhibitor MG-132 on HeLaM cells stably expressing VPS33A^WT-HA or VPS33A^R498W-HA, immunoblotted VPS33A bands were quantified by densitometry using ImageJ software, normalized to actin bands and fold changes in VPS33A concentration calculated relative to no incubation with MG-132. In each separate experiment, the effect of MG-132 on both VPS33A^WT-HA and VPS33A^R498W-HA was measured. A paired student t-test was used to calculate P-values.

The antibodies used were: Polyclonal rabbit anti‐VPS18 and polyclonal rabbit anti‐VPS33A [both as described 44], polyclonal rabbit anti‐Actin (A2066; Sigma‐Aldrich), monoclonal mouse anti‐HA (HA.11, MMS‐101R; Covance), polyclonal rabbit anti‐GFP 70, monoclonal mouse anti‐LC3 (4E12, M152‐3, MBL, used for immunofluorescence staining), polyclonal HRP conjugated goat‐anti‐rabbit IgG (A9169; Sigma‐Aldrich) and polyclonal HRP conjugated rabbit‐anti‐mouse IgG (A9044; Sigma‐Aldrich).