Anti rps6

Zebrafish tissues were lysed in 1× SDS sample buffer (50 mmol/L Tris-HCl pH 6.8, 2% SDS, 0.01% bromophenol blue, 10% glycerol, and 100 mmol/L dithiothreitol) and boiled at 98 °C for 10 min. Proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes, which were incubated overnight with primary antibodies (anti-p-S6, Cell Signaling Technology, Boston, USA, cat#4858S; anti-RPS6, Proteintech, Chicago, USA, cat#14823-1-AP; and anti-β-actin, Proteintech, cat#66009-1-Ig), washed three times, and incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibodies (anti-mouse IgG, Cell Signaling Technology, cat#7076S; and anti-rabbit IgG, Cell Signaling Technology,cat#7074S). Immuno-signals were generated by chemiluminescence (Cell Signaling Technology, cat#12757), scanned by a FluorChem E system (ProteinSimple, Silicon Valley, USA), and analyzed using ImageJ software.

HEK293T cells were seeded at approximately 30% confluence in a confocal dish and transfected with the indicated plasmids. Indirect immunofluorescence staining of transfected cells was performed at 48 h after transfection as described previously (38 (link)). Antihemagglutinin (anti-HA) (Sigma; h6908) and anti-RPS6 (Proteintech; 66886-1-1g) were used as the primary antibodies; Alexa Fluor 488-conjugated antibody and Alexa Fluor 568-conjugated antibodies (Life Technologies) were used as the secondary antibodies. The nuclei were stained with Hoechst 33258 (Life Technologies). The cells were then analyzed using a confocal laser scanning microscope (TCS SP8 STED; Leica, Germany).

HEK293T cells were seeded at approximately 30% confluence in a confocal dish and transfected with the indicated plasmids. Indirect immunofluorescence staining of transfected cells was performed at 48 h after transfection as described previously (38 (link)). Antihemagglutinin (anti-HA) (Sigma; h6908) and anti-RPS6 (Proteintech; 66886-1-1g) were used as the primary antibodies; Alexa Fluor 488-conjugated antibody and Alexa Fluor 568-conjugated antibodies (Life Technologies) were used as the secondary antibodies. The nuclei were stained with Hoechst 33258 (Life Technologies). The cells were then analyzed using a confocal laser scanning microscope (TCS SP8 STED; Leica, Germany).