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. 2021 Mar 30;65(6):630–645. doi: 10.1165/rcmb.2020-0537OC

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Figure 4. — Rat pulmonary microvascular endothelial cells (PMVECs) expressing domain-specific CA IX functional mutants confirm a unique role of the intracellular (IC) domain in mediating CA IX membrane localization. (A) The CA IX functional mutant-generation strategy is schematically illustrated. CA IX consists of four functional domains: a proteoglycan-like (PG) domain, a catalytic CA domain, a transmembrane (TM) domain, and an IC domain. (B) Double transfection with a doxycycline-induced Tet-off conditional expression system and domain-specific CA IX mutant constructs was performed on previously generated CA IX–K/O PMVECs. CA IX protein abundance was assessed and quantified in cell lysates by using Western blotting with the FLAG-tag antibody. CA IX functional mutant PMVECs were successfully generated indicated by CA IX monomer molecular weight shifts (arrows) and protein abundance decreases upon doxycycline introduction. (C) To further visualize CA IX mutant protein expression, we performed immunocytochemistry on these CA IX mutant cells by using an HRP (horseradish peroxidase) and 3,3'-diaminobenzidine detection method. Both M75 and FLAG tag antibody staining were consistent with the presence and absence of the epitope of each mutant cell line. Notably, ΔIC (IC domain-deleted) PMVECs showed a distinctive staining pattern in which the proteins are localized to perinuclear areas, saving intercellular borders. (D) N-cadherin membrane localization was unaffected by the absence of the IC domain, indicating that the mediating role of the IC domain in protein trafficking and membrane stabilization is specific to the CA IX protein. At least three separate experiments were performed. Scale bar, 400 μm (for C and D). Data represent the mean ± SD. One-way ANOVA and Bonferroni post hoc tests were used to compare different groups. *Significant difference (P < 0.05) from rescued wild-type (rWT) dose of 0 μg/ml doxycycline (B), rWT control IgG (C), and ΔIC control IgG (D). ΔCA = CA domain deleted; ΔPG = PG domain deleted; K/O = knockout; Tet-off = tetracycline off.

Figure 4. — Rat pulmonary microvascular endothelial cells (PMVECs) expressing domain-specific CA IX functional mutants confirm a unique role of the intracellular (IC) domain in mediating CA IX membrane localization. (A) The CA IX functional mutant-generation strategy is schematically illustrated. CA IX consists of four functional domains: a proteoglycan-like (PG) domain, a catalytic CA domain, a transmembrane (TM) domain, and an IC domain. (B) Double transfection with a doxycycline-induced Tet-off conditional expression system and domain-specific CA IX mutant constructs was performed on previously generated CA IX–K/O PMVECs. CA IX protein abundance was assessed and quantified in cell lysates by using Western blotting with the FLAG-tag antibody. CA IX functional mutant PMVECs were successfully generated indicated by CA IX monomer molecular weight shifts (arrows) and protein abundance decreases upon doxycycline introduction. (C) To further visualize CA IX mutant protein expression, we performed immunocytochemistry on these CA IX mutant cells by using an HRP (horseradish peroxidase) and 3,3'-diaminobenzidine detection method. Both M75 and FLAG tag antibody staining were consistent with the presence and absence of the epitope of each mutant cell line. Notably, ΔIC (IC domain-deleted) PMVECs showed a distinctive staining pattern in which the proteins are localized to perinuclear areas, saving intercellular borders. (D) N-cadherin membrane localization was unaffected by the absence of the IC domain, indicating that the mediating role of the IC domain in protein trafficking and membrane stabilization is specific to the CA IX protein. At least three separate experiments were performed. Scale bar, 400 μm (for C and D). Data represent the mean ± SD. One-way ANOVA and Bonferroni post hoc tests were used to compare different groups. *Significant difference (P < 0.05) from rescued wild-type (rWT) dose of 0 μg/ml doxycycline (B), rWT control IgG (C), and ΔIC control IgG (D). ΔCA = CA domain deleted; ΔPG = PG domain deleted; K/O = knockout; Tet-off = tetracycline off.