MYO1F interacts with TRIM21 to prevent the ubiquitination and degradation of PHB1. (A) Immunofluorescent staining of TRIM21 (red) and MYO1F (green) on neutrophils from WT BM obtained with laser modes and confocal fluorescence microscopy, DAPI used for cell nucleus indication. White arrowhead, colocalization of red and green colocalization (fosi). Scale bar: 50 µm. (B) Endogenous IP (co-IP) blot with BM neutrophils from WT mice, where IP was performed for MYO1F, and then precipitates were immunoblotted (IB) with an anti-TRIM21 antibody. (C) Exogenous co-IP blot with transfection of Flag-tagged MYO1F and His-tagged TRIM21 in 293T, where IP was performed for Flag, and then IB with an anti-His antibody. (D) Schematic diagram of protein structure of MYO1F and TRIM21. (E and F) Specific combined domain to TRIM21 on MYO1F. Exogenous co-IP blot with transfection of Flag-tagged TRIM21 and His-tagged truncated MYO1F in 293T, IP was performed for Flag, and then precipitates were IB with an anti-His antibody. (G and H) Specific combined domain to MYO1F on TRIM21. Exogenous Co-IP blot with transfection of Flag-tagged MYO1F and His-tagged TRIM21 in 293T, IP was performed for Flag, and then precipitates were IB with an anti-His. (I) MYO1F effect on TRIM21 and PHB1 binding ability. Exogenous co-IP blot with transfection of Flag-tagged TRIM21, His-tagged PHB1, and Myc-tagged MYO1F in 293T, IP was performed for Flag, and then precipitates were IB with anti-His and anti-Myc antibodies. (J) MYO1F effect on exogenous PHB1 ubiquitination. Exogenous co-IP blot with transfection of Flag-tagged PHB1, His-tagged TRIM21, Myc-tagged MYO1F, and HA-tagged ubiquitin in 293T, IP was performed for Flag, and then precipitates were IB with anti-ubiquitin antibody. (K) Cultured neutrophils from WT mice were transfected with siPhb1, and MYO1F protein levels were analyzed 48 h after transfection by immunoblotting. (L) Cultured neutrophils from WT mice were transfected with increased siMyo1f (0, 5, 10, and 20 pM); MYO1F and PHB1 protein levels were analyzed 48 h after transfection by immunoblotting. (M) MYO1F effect on endogenous PHB1 ubiquitination. Cultured neutrophils from WT mice were transfected with siMyo1f 20 pM, and PHB1 was immunoprecipitated with corresponding antibody and IB with anti-ubiquitin antibody. (N) Schematic diagram of MYO1F regulates STAT3 activation by interacting with TRIM21 and protecting PHB1 from degradation. Data in A–M represent one experiment of three independent repeats. Source data are available for this figure: SourceData F5.
Figure 5.

MYO1F interacts with TRIM21 to prevent the ubiquitination and degradation of PHB1. (A) Immunofluorescent staining of TRIM21 (red) and MYO1F (green) on neutrophils from WT BM obtained with laser modes and confocal fluorescence microscopy, DAPI used for cell nucleus indication. White arrowhead, colocalization of red and green colocalization (fosi). Scale bar: 50 µm. (B) Endogenous IP (co-IP) blot with BM neutrophils from WT mice, where IP was performed for MYO1F, and then precipitates were immunoblotted (IB) with an anti-TRIM21 antibody. (C) Exogenous co-IP blot with transfection of Flag-tagged MYO1F and His-tagged TRIM21 in 293T, where IP was performed for Flag, and then IB with an anti-His antibody. (D) Schematic diagram of protein structure of MYO1F and TRIM21. (E and F) Specific combined domain to TRIM21 on MYO1F. Exogenous co-IP blot with transfection of Flag-tagged TRIM21 and His-tagged truncated MYO1F in 293T, IP was performed for Flag, and then precipitates were IB with an anti-His antibody. (G and H) Specific combined domain to MYO1F on TRIM21. Exogenous Co-IP blot with transfection of Flag-tagged MYO1F and His-tagged TRIM21 in 293T, IP was performed for Flag, and then precipitates were IB with an anti-His. (I) MYO1F effect on TRIM21 and PHB1 binding ability. Exogenous co-IP blot with transfection of Flag-tagged TRIM21, His-tagged PHB1, and Myc-tagged MYO1F in 293T, IP was performed for Flag, and then precipitates were IB with anti-His and anti-Myc antibodies. (J) MYO1F effect on exogenous PHB1 ubiquitination. Exogenous co-IP blot with transfection of Flag-tagged PHB1, His-tagged TRIM21, Myc-tagged MYO1F, and HA-tagged ubiquitin in 293T, IP was performed for Flag, and then precipitates were IB with anti-ubiquitin antibody. (K) Cultured neutrophils from WT mice were transfected with siPhb1, and MYO1F protein levels were analyzed 48 h after transfection by immunoblotting. (L) Cultured neutrophils from WT mice were transfected with increased siMyo1f (0, 5, 10, and 20 pM); MYO1F and PHB1 protein levels were analyzed 48 h after transfection by immunoblotting. (M) MYO1F effect on endogenous PHB1 ubiquitination. Cultured neutrophils from WT mice were transfected with siMyo1f 20 pM, and PHB1 was immunoprecipitated with corresponding antibody and IB with anti-ubiquitin antibody. (N) Schematic diagram of MYO1F regulates STAT3 activation by interacting with TRIM21 and protecting PHB1 from degradation. Data in A–M represent one experiment of three independent repeats. Source data are available for this figure: SourceData F5.

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