Cr7699 4

Protein samples were isolated from cultured mouse mesothelial cells and from mouse tissue. Cells were grown in 25 cm² flasks and harvested at confluence. Cytosolic protein fractions were collected as described before [15 (link)]. Freshly excised mouse cerebellum was frozen in liquid nitrogen and homogenized in extraction buffer (10 mM Tris, 2 mM EDTA, 1 mM β-mercaptoethanol; pH 7.4) containing a cocktail of different protease inhibitors (Roche, Mannheim, Germany). Proteins (100 μg) from each cell culture sample, 1 μg protein from cerebellum, as well as 40 ng of purified human recombinant CR were loaded onto SDS-polyacrylamide gels (10 %). After separation, proteins were blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated for two days at 4 °C with the CR-specific antibody CR7699/4 (Swant, Marly, Switzerland) at a dilution of 1:10,000. Rabbit secondary antibody linked to horseradish peroxidase (Sigma-Aldrich) was diluted at 1:10,000 and incubated for 2 days; prolonged incubation was shown to enhance the sensitivity in Western blotting [16 (link)]. For the detection, the chemiluminescent reagent Luminata Classico Forte (EMD Millipore Corporation, Billerica, MA, USA) was used. Chemiluminescent and normal illumination digital images were recorded on a system from Cell Biosciences (Santa Clara, CA, USA).

MM cells at a confluence of 70–90% were washed with ice-cold PBS and lysed with 1 ml ice-cold Triton X-100 lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0) supplemented with protease and phosphatase inhibitors. Cell lysates were incubated on ice for 30 min and then centrifuged (10,000×g, 10 min at 4 °C). To pull down CR, 2–4 μg of a rabbit polyclonal anti-CR antiserum (CR7699/4; Swant) was added to the cleared lysate and incubated for 10 min at 4 °C. μMACS protein A MicroBeads suspension (100 μl; Miltenyi Biotec, Auburn, CA, USA) was added to the lysate and incubated at 4 °C for 30 min. Samples were loaded on MACS separation columns (Miltenyi Biotec) and subjected to magnetic immunoprecipitation. The columns were washed 3 times with a wash buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0). Bound protein complexes were eluted in 50 μl of pre-warmed SDS gel loading buffer 1X (50 mM Tris-HCl, pH 6.8, 50 mM DTT, 1% SDS, 0.005% bromophenol blue, 10% glycerol) and subjected to Western Blotting for septin 7 as described above. A mouse monoclonal anti-Paxillin antibody (1:2000; BD Bioscience) was used as negative control.

pLV-CALB2 lentiviral vectors were used to stably express CR in different cell lines. Cells were tested for CR expression by Western Blot analysis using the CR antibody CR 7699/4 (Swant, Marly, Switzerland). MSTO-211H cells were infected with a lentivirus carrying the hRluc reporter gene. After each passage, cells were lysed and the stable and long-lasting expression of Renilla luciferase was measured using Renilla Juice (p.j.k.-GmbH, Kleinblittersdorf, Germany) in a Turner Designs TD-20/20 Luminometer (Sunnyvale, CA, USA) according to the manufacturer’s protocol.