Rabbit anti mcpip1

Cell lysates were separated by SDS/PAGE (8, 10 or 12% polyacrylamide gel), electrotransfered to PVDF membrane (Millipore,) and blocked for 1 h (at room temperature) in 5% non-fat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (BioShop, Burlington, Canada). Membranes were incubated with primary antibody overnight at 4 °C. After incubation with secondary antibody for 1 h, room temperature, chemiluminescence was detected using Immobilon Western HRP substrate (Millipore) with ChemiDoc system (BioRad). The following antibodies and dilutions were used: rabbit anti-MCPIP1 (1:1000, GeneTex), HIF1α (1:1000), HIF2α (1:1000), VHL (1:500), Akt (1:1000), Phospho-Akt (1:2000), SAPK/JNK (1:500), Phospho-SAPK/JNK (1:1000), p38 (1:1000), Phospho-p38 (1:1000), ERK1/2 (1:1000), Phospho-ERK1/2 (1:1000). All mentioned antibodies were from Cell Signaling Technology. Tubulin (1:4000, Calbiochem; Merck Millipore, Billerica, MA, USA) or in case tissue samples, GAPDH (1:30,000 Sigma-Aldrich) antibodies were used as a loading control. The following secondary antibodies were used: peroxidase-conjugated anti-rabbit IgG (1:30,000; Sigma) and peroxidase-conjugated anti-mouse IgG (1:10,000, Sigma).

Slides with gingival tissue or with TIGK cells were blocked with PBS containing 5% FBS, 1% BSA, 0.05% Tween 20, and 2 mM EDTA for 1 h at room temperature (RT). Slides were treated with 0.1% saponin in PBS for 30 min at RT and stained with primary antibodies diluted in buffer (3% BSA and 0.1% saponin in PBS). TIGK cells were stained for 1 h at RT with mouse anti-MCPIP-1 (10 μg/ml; R&D) and rabbit anti-RgpA (10 μg/ml) antibodies; gingival tissues were stained with rabbit anti-Mcpip-1 (13.5 μg/ml; GeneTex). Slides were washed with 0.1% saponin in PBS and stained with secondary antibodies for 45 min at RT. All slides were incubated with goat anti-rabbit antibodies conjugated with Alexa 488 (1:500; Cell Signaling); in addition, TIGK cells were stained with goat anti-mouse antibodies conjugated with Alexa 647 (1:500; Cell Signaling). After several washes with 0.1% saponin in PBS, cell nuclei were stained with Hoechst 33342 (1 μg/ml) for 10 min at RT. Then slides were washed in PBS and mounted in fluorescence medium (Dako). Images were captured with a confocal laser scanning microscope (LSM 880; Zeiss). Quantification of fluorescence signal was with ImageJ software and is displayed as corrected total cell fluorescence (CTCF) or as a percentage of area for fluorescence signal.