Anti his hrp antibody

Antibodies against YAP and HA-F7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–FLAG-M2 and β-actin antibodies were from Sigma Aldrich. TAZ antibody was from BD Transduction Laboratories. Anti-His-HRP antibody was obtained from Miltenyi Biotec Inc. Restriction enzymes for cloning were purchased from NEB (New England Bio lab). HisTrap^™ and GSTrap^™ Fast Flow columns were from GE Healthcare. Protein G/A-Agarose was purchased from Roche Diagonistics.

For SDS-PAGE analysis, 100 µg denatured protein of the bacterial lysate supernatants were run on a 7.5% polyacrylamide gel. The proteins were then transferred onto a Protran BA 85 Membrane (Carl Roth GmbH, Karlsruhe, Germany) and the blots were probed with an anti-His-HRP antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Immunoreactive bands were visualized using the SERVALight Polaris CL HRP WB Substrate Kit (Serva Electrophoresis GmbH, Heidelberg, Germany). Chemiluminescence was detected on a FUJIFILM Luminescent Image Analyzer LAS-1000plus & Intelligent Dark Box II. The protein concentrations in the bacterial lysates were quantified using Bradford Reagent for quantitative protein determination (AppliChem, VWR International GmbH, Darmstadt, Germany) according to the instructions of the vendor. Amino acid sequence alignments were performed using the EMBOSS Needle tool (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ accessed on 25 April 2019).

To test ALOX15 expression aliquots (2–20 µL) of the lysis supernatants were analyzed by SDS-PAGE on a 7.5% polyacrylamide gel. Separated proteins were transferred to a 0.45 µm nitrocellulose membrane (Thermo Scientific GmbH, Schwerte, Germany) by a wet blotting method (ProSieve Ex Western Blot Transfer Buffer 10x, Biozym Scientific GmbH, Hessisch-Oldendorf, Germany). The membranes were blocked with blocking solution (10x BlueBlock PF for Blotting, SERVA Electrophoresis GmbH, Heidelberg, Germany), washed three times with PBS/0.3% TWEEN 20 and were finally incubated with an anti-His-HRP antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for 1–2 h at room temperature. After several steps of washing, the membrane was stained using the SERVALight Polaris CL HRP WB Substrate Kit (SERVA Electrophoresis GmbH, Heidelberg, Germany) for 5 min at room temperature. Chemiluminescence was quantified using the FUJIFILM Luminescent Image Analyzer LAS-1000plus (Fujifilm Europe GmbH, Düsseldorf, Germany).