Wb stripping solution

Immunoblot analysis was performed as previously described [19 (link)] with primary antibodies to phosphorylated Akt (#4060), to Akt (#9272), to phosphorylated ERK1/2 (#4370), to ERK1/2 (#9102), to phosphorylated MEK (#9121), to MEK (#9122), to phosphorylated S6 (#4858), to S6 (#2217), to phosphorylated STAT3 (#9131), to STAT3 (#4904), and to SHP-2 (#3752), all of which were obtained from Cell Signaling Technology, as well as with those to BRAP (sc-166012), to neurofibromin (sc-376886), and to α-tubulin (sc-32293) from Santa Cruz Biotechnology. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare and Dako), enhanced chemiluminescence reagents (ImmunoStar LD, Wako), and a LAS-3000mini instrument (GE Healthcare, Chicago, IL, USA). After the detection of the phosphorylated forms of Akt, ERK1/2, MEK, S6, and STAT3, the membrane was stripped with WB Stripping Solution (Nacalai Tesque) and then reprobed with antibodies to the corresponding total forms of these proteins.

Proteins in PPP-VF (10 µl), and in GST-hCAP18 recombinant protein preparations after incubation with various factors, were resolved on 15% (w/v) Tris-HCl gels (BIO CRAFT, Tokyo, Japan) and transferred to PVDF membranes (Immobilon-P; Millipore, Darmstadt, Germany); 3.2 pmol of the LL-37 synthetic peptide served as a positive control. Membrane bands were visualized as described above. To confirm the identities of bands detected by Western blotting, the filter was stripped of antibody using WB Stripping Solution (Nacalai Tesque, Kyoto, Japan) following the manufacturer’s instructions, and next reacted with a chicken anti-cathelin polyclonal antibody (1∶2,000) and an HRP-conjugated-anti-chicken IgY goat antibody (1∶2,000, Promega), as described previously [30] (link).
To detect proteinase 3 in PPP-VF, 10-µl PPP-VF were subjected to 15% (w/v) Tris-HCl gel electrophoresis using native human proteinase 3 as a positive control. A mouse anti-proteinase 3 monoclonal antibody (1∶30) was used as primary antibody to detect the protein, as described above.

The cells were lysed with radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Nacalai). Following centrifugation, the supernatant containing the total proteins was fractionated by sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (TEFCO, Hachioji, Tokyo, Japan). The separated proteins were transferred to polyvinylidene difluoride membranes (TEFCO), blocked with 5% of BlockingOne (Nacalai) for 30 min, and incubated with anti-HA (Cell Signaling Technology; diluted 1/1000), anti-HistonH3 (Cell Signaling Technology; diluted 1/1000), anti-CD9 (WAKO; diluted 1/500), and anti-CD81 (WAKO; diluted 1/500) primary antibodies overnight at 4 °C. The blots were probed with horseradish peroxidase-conjugated secondary antibodies (Molecular Probes; diluted 1/5000) and developed with luminal for enhanced chemiluminescence using Chemi-Lumi One Super (Nacalai). When probing for multiple targets, a single membrane was stripped with WB Stripping Solution (Nacalai) and re-probed with antibodies again.

Lentivirally transduced J.CaM2 cells were starved in RPMI 1640 without FCS for 3 h before being stimulated with anti-CD3 mAb at 37 °C. Cells were then lysed at 2.0 × 10⁷ cells/mL in 2× Laemmli buffer, followed by incubation at 99 °C for 5 min and sonication. For Western blotting, whole-cell lysates were separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with the indicated primary antibodies, followed by the appropriate secondary antibody conjugated to IRDye 800 CW (Li-Cor, Lincoln, NE, USA) or horseradish peroxidase (HRP). Reactive proteins were visualized using the Odyssey CLx Infrared Imaging System (Li-Cor) or by enhanced chemiluminescence (ECL) acquired in a ChemiDoc Touch Imaging System (Bio-Rad Laboratories). For reprobing, PVDF membranes were incubated for 10 min at room temperature with WB Stripping Solution (Nacalai Tesque, Kyoto, Japan), followed by a TTBS wash.

Lentivirally transduced Jurkat cells were starved in RPMI 1640 without FCS for 3 h prior to stimulation with anti-CD3 monoclonal antibody (mAb) at 37 °C. Cells were then lysed at 2.0 × 10⁷ cells/mL in 2× Laemmli buffer, followed by incubation at 99 °C for 5 min and sonication. For Western blotting, whole-cell lysates were separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with the indicated primary antibodies, followed by the appropriate secondary antibody conjugated to IRDye 800 CW (Li-Cor, Lincoln, NE, USA) or horseradish peroxidase (HRP). Reactive proteins were visualized using the Odyssey CLx Infrared Imaging System (Li-Cor, Lincoln, Nebraska USA), or by enhanced chemiluminescence (ECL) acquired in a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). For reprobing, PVDF membranes were incubated for 10 min at room temperature with WB Stripping Solution (Nacalai Tesque, Kyoto, Japan), followed by a TTBS wash.

Lentivirally transduced J.CaM2 cells were starved in RPMI 1640 without FCS for 18 h before being stimulated with anti-CD3 mAb at 37°C. Cells were then lysed at 2.5 × 10⁷ cells/ml in 2× Laemmli buffer, followed by incubation at 99°C for 5 min and sonication. For anti-Fas stimulation, cells were incubated with 100 ng/ml of anti-Fas mAb at 1 × 10⁶ cells/ml in RPMI 1640, supplemented with 10% FCS, and then pelleted and lysed as described earlier. For Western blotting, whole-cell lysates were separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with the indicated primary antibodies, followed by the appropriate secondary antibody conjugated to IRDye 800CW (Li-Cor, Lincoln, NE, USA) or horseradish peroxidase (HRP). Reactive proteins were visualized using the Odyssey CLx Infrared Imaging System (Li-Cor) or by enhanced chemiluminescence (ECL) acquired in a ChemiDoc Touch Imaging System (Bio-Rad Laboratories). For reprobing, PVDF membranes were incubated for 10 min at room temperature with WB Stripping Solution (Nacalai Tesque, Kyoto, Japan), followed by a TTBS wash. For the cycloheximide chase assay, cells were treated with 0.1 mM cycloheximide for up to 10 h. Every 2 h, cell samples were lysed in 2× Laemmli buffer and LAT protein levels were determined by immunoblotting and quantified by densitometry.