Hrp conjugated goat anti rabbit igg

To determine the inhibition of rTs-Pmy on the binding of C1q to IgM, the ELISA assay was performed. Plates were coated with 2 μg/ml of human IgM in 100 μl of coating buffer (100 mM Na2CO3/NaHCO3, pH 9.6) at 4°C overnight. After washing three times with PBST, the plates were blocked with 1 × PBS containing 2% BSA for 2 h at 37°C. One μg of C1q was pre-incubated with different amounts of rTs-Pmy or BSA (0, 2, 3, 4 μg) for 2 h at 37°C, then added to the plates coated with IgM for 1 h at 37°C. After washing three times with PBST, the binding of C1q to IgM was determined with anti-C1q polyclonal antibody (Abcam, USA; 1:3,000 in 1% BSA/PBS). HRP-conjugated goat anti-rabbit IgG (BD Biosciences, USA) was used as the secondary antibody and OPD (Sigma, USA) was used as the substrate. The absorbance of the supernatants was measured at 450 nm with a MultiskanGO plate reader (Thermo, USA).

To evaluate whether the binding of rTs-Pmy to C1q inhibited complement activation, C3 deposition following complement activation were analyzed [23 (link)]. Plates were coated with 2 μg/ml of human IgM in 100 μl of coating buffer (100 mM Na2CO3/NaHCO3, pH 9.6) at 4°C overnight. After washing three times with PBST, the plates were blocked with 1 × PBS containing 5% BSA for 2 h at 37°C. Two μg of C1q was pre-incubated with different amounts of rTs-Pmy (0, 2, 4 μg) and BSA (4 μg, as a control) for 2 h at 37°C before adding to the plates coated with IgM (the activator) for 1 h at 37°C. After washing three times with PBST, C1q-deficient serum (C1q D) diluted to 2% in GVBS⁺⁺ (Veronal-buffered saline containing 1 mM MgCl2, 0.15 mM CaCl2, 0.05% Tween-20, and 0.1% gelatin, pH 7.4) was added as a source of rest complement components to the plates for 1 h at 37°C and then washed with PBST three times. C3 deposition was determined with anti-C3 polyclonal antibody (Abcam, USA; 1:5,000 in 1% BSA/PBS). HRP-conjugated goat anti-rabbit IgG (BD Biosciences, USA) was used as the secondary antibody and OPD (Sigma, USA) was used as the substrate. The absorbance of the supernatants was measured at 450 nm with a MultiskanGO plate reader (Thermo, USA).

The following primary antibodies were used: Rabbit anti-SPBP antibody [27] (link), monoclonal anti-p62 antibody (BD Transduction Laboratories), rabbit anti-NRF2 antibody (Santa Cruz, SC-13032, Abcam ab62352), rabbit anti-LC3B antibody (SIGMA, L7543), rabbit anti-KEAP1 antibody (ProteinTech, 10503-2-AP), mouse anti-Flag antibody (Stratagene, 200471), rabbit anti-actin antibodies (Sigma, A 2066). Secondary antibodies used were: HRP-conjugated goat anti-rabbit IgG and anti-mouse IgG antibodies (BD Bioscience Pharmingen). AlexaFluor 568 and AlexaFluor 548 conjugated goat anti mouse and anti-rabbit IgG (Molecular Probes). Sulforaphane (S 4441), Bafilomycin A1 (B 1793) and MG 132 (C 2211) were purchased from Sigma-Aldrich.

Protein expression levels were determined by western blot analysis, as previously described [61 (link)]. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary antibodies against MMP-2 (Cell Signaling #4022), MMP-9 (Cell Signaling #13667), total PI3K (Cell Signaling #4292), phospho-PI3K (Cell Signaling #4228), total Akt (Cell Signaling #4491), phospho-Akt (Cell Signaling #4060), total-ERK1/2 (Cell Signaling #9012), phospho-ERK1/2 (Cell Signaling #9101), total FAK (Santa Cruz, sc-558), phospho-FAK (Santa Cruz, sc-11765), or β-actin (Abcam, ab189073) overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, 554021) or goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using enhanced chemiluminescence (ECL) reagents.

The protein expression levels were determined by western blot analysis as previously described^{53 (link)}. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary antibodies against β-actin (Abcam, MA, USA, ab189073), MMP-2 (Cell signaling #4022), MMP-9 (Cell Signaling #13667), total PI3K (Cell Signaling #4292), phospho-PI3K (Cell Signaling #4228), total Akt (Cell Signaling #4491), phospho-Akt (Cell Signaling #4060) overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, USA, 554021) and goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using ECL reagents.

Protein expression levels were determined by western blot analysis as previously described^{23 (link)}. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations in the total cell lysates were measured by using bovine serum albumin as the standard. Samples containing equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), and proteins were then transferred onto nitrocellulose membranes (Bio–Rad Laboratories). Membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 at RT. Then, the membranes were incubated first with primary antibodies against β-actin (Abcam, MA, USA, ab189073), MMP-2 (Cell Signaling #4022), MMP-9 (Cell Signaling #13667), SERPINB2 (Abcam, MA, USA, ab47742), and caspase-3 (Cell Signaling, MA, USA, #9662) overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, USA, 554021) and goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using ECL reagents.

The protein expression levels were determined by western blot analysis as previously described [36 (link)]. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40 and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary anti-SERPINB2 (Abcam, MA, USA, ab47742) and β-actin (Abcam, MA, USA, ab189073) antibodies overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, USA, 554021) and HRP goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using an ECL reagent.