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Ha 11mms 101r

Manufactured by Fortrea

The HA-11MMS-101R is a laboratory equipment designed for high-precision measurements. It features an advanced microprocessor-based system that provides accurate and reliable data acquisition. The core function of this product is to facilitate precise measurement and data analysis in various scientific and industrial applications.

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3 protocols using ha 11mms 101r

1

Western Blotting Protocol for Wnt Signaling

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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.

Antibodies

AntibodyManufacturerDilution and applicationReference
LRP6 (C47E12)cs-3395, Cell Signaling Technology1:1000, WB[28 ]
LRP6 (Phospho-S1490)cs-2568, Cell Signaling Technology1:1000, WB[28 ]
β-actinCS-4970, Cell Signaling Technology1:3000, WB[26 (link)]
DVL-2CS-3216, Cell Signaling Technology1:1000, WB[26 (link)]
DVL-3CS-3218, Cell Signaling Technology1:1000, WB[26 (link)]
HA-11MMS-101R, Covance1:2000 WB; 1:500, IF[28 ]
a-mouse IgG HRPA44161:4000 WB
a-rabbit IgG HRPA05451:4000 WB
a-mouse Alexa Fluor™568A10037, Thermo Fisher Scientific1:600 IF
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2

VPS33A Proteasomal Regulation in Cells

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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 VPS33AWT-HA or VPS33AR498W-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 VPS33AWT-HA and VPS33AR498W-HA was measured. A paired student t-test was used to calculate P-values.
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3

Antibody Characterization for Subcellular Localization

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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).
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