Ab11433

For immunofluorescence studies, chicken anti-human/mouse RGS5 antibody was used (GW22900, Sigma Aldrich^®, Schnelldorf, Germany). For capillary electrophoresis, the anti-human/mouse RGS5 antibody (BYT-ORB6873, Biozol, Eching, Germany) was utilized to detect RGS5. The rabbit anti-human Ki67 antibody used for quantification of cell proliferation was purchased from Abcam (ab16667, Cambridge, UK). The mouse anti-human/mouse/rat RhoA (26C4) antibody (sc-418) and the mouse anti-recombinant His-6 antibody (sc-8036) were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). The mouse anti-human VCP antibody (ab11433, Cambridge, UK) was purchased from Abcam. Antibodies against total ERK1/2 (#4695) as well as phosphorylated ERK1/2 (#4370) were purchased from Cell Signaling Technology^® (Danvers, MA, USA). Pertussis Toxin (#3097) was purchased from Tocris (Bristol, UK) and YM-25490 (#257-00631) from Fujifilm Wako Chemicals Europe GmbH (Neuss, Germany).

Cells were harvested in protein lysis buffer consisting of 25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 0.5% Triton-X100, 0.5% NP-40, 1x proteinase inhibitor cocktail (PIC) (Sigma), 0.5 mM Na₃VO₄, 10 mM NaF and 10 mM C₃H₇Na₂O₆P. Protein concentration was quantified using the BCA Protein Assay (Pierce) and 20 µg of protein were loaded to 10% SDS-polyacrylamide gels and blotted to nitrocellulose membranes. The antibodies used for Western Blot were specific for thymoma viral proto-oncogene 1 (AKT), p-AKT (9272 and 9271, Cell Signaling), GLUT-1 (abcam ab32551), and VCP (ab11433, Abcam) for normalizing. Quantification was performed using the ImageJ software.

The various tissues were fixed in 10% neutral buffered formalin and afterwards embedded in paraffin. For immunohistochemistry, tissue sections (4 μm thickness) were deparaffinized, rehydrated and subjected to heat-induced antigen retrieval in Tris-EDTA buffer (10mM Tris, 1mM EDTA, pH 9). The sections were permeabilized with 0.5% Tween 20 in PBS for 15 min at room temperature and blocked with 5% BSA, 5% goat serum in PBST (0.05% Tween) for 90 minutes at room temperature. Slides were then incubated with the primary antibodies overnight at 4°C. After 3 washes with PBST for 5 min, the sections were incubated in the dark with the secondary antibodies. For counterstain DAPI (1 μg/ml) was used and Mowiol 4–88 (Polysciences) was used for mounting. Primary antibodies: mouse anti-VCP (1:250, Abcam, ab11433), rabbit polyclonal anti-K315me3 (1:500, New England Peptides, custom antibody against synthetic peptide H2N-AIAPKRE(3me)KTHGEVERR-OH, double affinity purified), anti-VCPKMT (1:500, serum of rabbits immunized with recombinant human VCPKMT [6 (link)]). Secondary antibodies: goat anti-rabbit-Alexa 488 (1:500, Life Technologies), goat anti-mouse-Alexa 594 (1:500, Life Technologies). Images of fluorescently stained sections were acquired using AxioCam MRRev3 camera on an Axio Observer. Z1 microscope (Carl Zeiss).

Cells were lysed with Cell Lysis Buffer (9803S, Cell Signaling Technology) containing 1 mM PMSF. Whole tissue lysates were generated using bead mills with 100 µl lysis buffer per 50 mg of tissue. SDS–PAGE was employed to separate proteins which were then blotted on a nitrocellulose membrane. Membranes were blocked in 5% skim milk dissolve in TBS with 0.1% Tween 20. Membranes were incubated with primary antibodies diluted in 5% skim milk in TBST overnight at 4°C. Following primary antibodies were used: anti‐β‐Actin (A5441, Sigma‐Aldrich, 1:3,000), anti‐BDH1 (15417‐1‐AP, Proteintech for Fig 1B, 1:2,000 or ab193156, Abcam, 1:1,000 for all other Western blots), anti‐SCOT (12175‐1‐AP, Proteintech, 1:1,000), anti‐β‐Tubulin (2146, Cell Signaling Technology, 1:2,500), anti‐VCP (ab11433, Abcam, 1:2,000). HRP‐conjugated secondary antibodies (DAKO) were used and chemiluminescence was detected by Aceglow ECL Western Blotting Substrate (VWR International).