Horseradish peroxidase link secondary antibodies

Generally 1.5 × 106 EAHY cells were inoculated onto 10-cm culture dishes and exposed to various concentrations of 7-KC or DMSO for 24h. Cell lysates were prepared by dissolving cells in lysis buffer (10 mM Tris-HCl, pH 7; 140 mM sodium chloride; 3 mM magnesium chloride; 0.5% NP-40; 2 mM phenylmethylsulfonyl fluoride; 1% aprotinin; and 5 mM dithiothreitol) and the same amounts of proteins (20-50 μg/ml) were loaded to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blotted first with primary antibodies against Cdk1, cyclin B1 and GAPDH for 2 hr as described previously [36 (link), 37 (link)]. This was followed by incubation with respective horseradish peroxidase-link secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hr. After rinsed the membrane with buffer, Enhanced chemiluminescence (ECL) reagents (Amersham, Piscataway, NJ, USA) were added and the chemiluminescence was detected by exposure of membranes to Fuji films for 30 sec to 10 min. The intensity of GAPDH bands was used as control.

Generally 1.5 × 10⁶ EAHY were inoculated onto 10-cm culture dishes and exposed to various concentrations of LPC for 24h. After removal of medium and washed with PBS, cell lysates were prepared by dissolving cells in lysis buffer (10 mM Tris-HCl, pH 7; 140 mM sodium chloride; 3 mM magnesium chloride; 0.5% NP-40; 2 mM phenylmethylsulfonyl fluoride; 1% aprotinin; and 5 mM dithiothreitol). Then the equal amounts of proteins (20–50 μg/ml) were loaded to run 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were first blotted with primary antibodies of cdc2, cyclin B1, IL-8 and GAPDH for 2 hr as described before [35 (link), 36 (link)]. The membranes were then incubated in respective horseradish peroxidase-link secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hr. After washing the membrane with buffer, ECL reagents (Amersham) were added and the chemiluminescence of protein bands was determined by exposure of membranes to Fuji films for 30 sec to 10 min. The intensity of GAPDH bands was used as control.

Briefly, 1.5 x 10⁶ human dental pulp cells were seeded into 10-cm culture dishes with 10 ml of DMEM with 10% FBS. After 24 hours, the medium was replaced by a fresh one containing DMSO (vehicle control, NC) or different concentrations of CQ (0.1, 0.25, 0.5, 1, and 2 mM). After 24 hours, cells were washed with PBS and then disrupted in lysis buffer (10 mM Tris-HCl, pH 7; 140 mM sodium chloride; 3 mM magnesium chloride; 0.5% NP-40; 2 mM phenylmethylsulfonyl fluoride; 1% aprotinin; and 5 mM dithiothreitol) [13 (link),23 (link)]. Then aliquots (20–50 μg protein) of cell lysates were loaded to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blotted first with anti-human HO-1, cdc2, cdc25c, cyclin B1, COX-2, p-ERK1/ERK2 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibodies for 2-hr. This was followed by incubation with horseradish peroxidase-link secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hr. After washing the membrane with buffer, ECL reagents (Amersham) were added and the chemiluminescence was detected by exposure of membranes to Fuji films for 30 sec to 10 min. The intensity of GAPDH was used as control.