Western Blot Analysis of C. elegans Proteins

Worms were lysed in 1x NuPAGE LDS sample buffer (ThermoFisher Scientific) and heated at 90 °C for 10 min. Any debris was removed by centrifuging at 18,000 × g. ~50 µg of protein extracts was then resolved on precast NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen, NP0321BOX). The proteins were transferred to a nylon membrane with the semidry transfer Pierce Power System (ThermoFisher Scientific) using the pre-programmed method for high-molecular-mass protein. The primary antibodies used included anti-KLP-7^{25 (link)} (1:1000 dilution) (a gift from the Desai laboratory), anti-tubulin (Ab6160, Abcam) (1:1000 dilution), anti-GAPDH (Ab125247, Abcam) (1:2000 dilution), anti-PGL-1^{69 (link)} (1:2000 dilution) (a gift from the Strome laboratory), anti-PRG-1^{70 (link)} (1:2000 dilution) (a gift from the Mello laboratory), anti-Flag (F3165, Sigma) (1:1000 dilution), anti-RPS-3 (ab128995, Abcam) (1:3000 dilution) and the secondary antibodies used included anti-rabbit (31460, Pierce) (1:10000 dilution), anti-mouse (31430, Pierce) (1:10000 dilution) and anti-rat (A9037, Sigma) (1:10000 dilution) HPR antibodies. The SuperSignal West Pico PLUS Chemiluminescent Substrate was used to detect the signal using a ChemiDoc MP imaging system (Biorad).