Fig. 2: KTN1 regulates EGFR degradation via the UPS.
A A431 cells were transfected with siNC or siKTN1 and collected after transfection for the indicated time periods. Total cell lysates were analyzed by immunoblotting with anti-KTN1 and anti-EGFR antibodies. Representative of results from three biological replicates. B Ten micromolar MG132 was added to siNC-transfected or siKTN1-transfected A431 cells, which were collected after transfection for the indicated time periods. Total cell lysates were analyzed by immunoblotting with anti-KTN1 and anti-EGFR antibodies. Representative of results from three biological replicates. C–F Gray value was analyzed by image J and to calculate Relative KTN1 or EGFR protein level. Quantified gray values represent the means ± SD. Representative of results from three biological replicates. G Protease activity assay of siNC-transfected cells, siKTN1-transfected cells or co-transfected with KTN1mut without or with MG132 treated for the indicated time periods. The results of proteasome activity represent the means ± SD. Representative of results from three biological replicates. H–J A431 cells were transfected with siNC or siKTN1 and collected at the indicated times. Total cell lysates were analyzed by Co-IP with anti-EGFR antibody for IP and anti-poly-ubiquitin antibody for immunoblotting. Quantified gray values represent the means ± SD. Representative of results from three biological replicates. K A431 cells were transfected with siNC or siKTN1 and collected at the indicated times. Total cell lysates were analyzed by immunoblotting with anti-UCH37 antibody. Representative of results from three biological replicates. L, M Gray value was analyzed by image J and to calculate Relative KTN1 or UCH37 protein level. Quantified gray values represent the means ± SD. Representative of results from three biological replicates. N A431 cells were transfected with siNC, siUCH37, and siUCH37#. Total cell lysates were analyzed by immunoblotting with anti-UCH37, anti-EGFR, and anti-poly-ubiquitin antibodies. Representative of results from three biological replicates. O A431 cells were transfected with siKTN1 and/or siUCH37. Total cell lysates were subjected to immunoblotting with anti-KTN1, anti-KTN1, and anti-poly-ubiquitin antibodies. Representative of results from three biological replicates.
