Role of SQSTM1 in regulation of ligand-induced CSF3R routing, stability and signaling. (A) HeLa-CSF3R cells were transfected with siRNAs against Sqstm1 or control siRNAs. Western blotting showed a ∼ 75% knock-down (KD) in cells transfected with Sqstm1 siRNAs compared with cells transfected with control siRNAs. (B) Transfected cells were stimulated with Bio-CSF3 or left unstimulated for 10 minutes at RT (t = 0), washed with RPMI and transferred to 37°C for indicated time points. CSF3R proteins were pulled down and analyzed as described in panel A. The ratio of detected CSFR protein levels siSQSTM1/siControl samples (n = 3) was calculated for 15 and 120 minutes CSF3 treatment and is depicted. The significance of the difference between the calculated ratios of time point 120 minutes and 15 minutes was calculated with the Mann-Whitney test (asymptotic significance [2-tailed]; P < .10). (C) HeLa-CSF3R cells were transfected with siRNAs against Sqstm1 (+) or control siRNAs (−). Western blotting showed a > 95% knockdown (KD) in cells transfected with siRNAs targeting Sqstm1 transcripts compared with cells transfected with control siRNAs. Cells were serum starved for 24 hours followed by CSF3 (100 ng/mL) treatment as described in panel A. Samples for cell lysates were taken at indicated time points and analyzed by Western blotting with total and/or phospho-specific antibodies against ERK.