Ure had no major effect on GPIanchored CD59. We previously reported a related observation for GPIanchored DAF expressionCurr. Issues Mol. Biol. 2021, 1, FOR PEER Review Curr. Problems Mol. Biol. 2021, 1, FOR PEER REVIEW4Curr. Concerns Mol. Biol. 2021,PLC) at a concentration (0.8 U/mL) sufficient to cleave the GPI anchor. Western blot evaluation of glomerular lysates revealed almost comprehensive absence of CD59 within the Recombinant?Proteins Testican 3 Protein presence of PLC) at a concentration (0.8 U/mL) sufficient to cleave the GPI anchor. Western blot1084 analPIPLC (Figure 1a) indicating that isolation process had no significant effect on GPIanysis of glomerular lysates revealed practically full absence of CD59 in the presence of chored CD59. We previously reported a comparable observation for GPIanchored DAF exPIPLC (Figure 1a) indicating that isolation process had no important effect on GPIanpression in isolated glomeruli [11]. For that reason, these experiments weren’t repeated. In CD59. comparable observation for GPIanchored DAF exin chored PIPLCWe previously reported aon the nonGPIanchored protein, Crry (Figisolated contrast, glomeruli [11]. Thus, these experiments were not repeated. In contrast, remedy had no effect pression in isolated no impact on[11]. nonGPIanchored protein, Crry (Figure 1b). glomeruli the Consequently, these experiments were not repeated. In PIPLC treatment had ure1b). contrast, PIPLC remedy had no effect around the nonGPIanchored protein, Crry (Figure1b).Figure 1. Detection of glomerular CD59 and Crry. Glomeruli had been incubated and phosphatidylinositolspecific phospho with Figure 1. Detection of glomerular CD59 with Crry. Glomeruli have been incubated lipase C (PIPLC) (0.eight U/mL) for 90 min to confirm the presence of GPIanchored CD59 protein in isolated glomeruli and phosphatidylinositolspecific phospholipase C (PIPLC) (0.eight U/mL) for 90 min to confirm the presFigure of membrane glomerular Total protein lysates have been analyzed by Western blot for CD59 and Crry protein. presence 1. Detection of bound Crry.CD59 and Crry. Glomeruli were incubated with phosphatidylinositolspecific phosphoence of GPIanchored CD59 protein in isolated glomeruli and presence of membrane bound Crry. lipase C (PIPLC) (0.eight U/mL) for 90 actin was applied as a loading handle. min to confirm the presence of GPIanchored CD59 protein in isolated glomeruli and presence of membrane bound protein lysates werelysates have been analyzedblotWestern blot andCD59 and Crry protein. Total Crry. Total protein analyzed by Western by for (a) CD59 for (b) Crry protein. actin was actin was employed as a loading control. usedHPXloading handle. Increases Glomerular HO1 Expression three.2. as a Deficient Serum To establish Serum Increases Glomerular HO1 Expression three.2. HPX Deficient Serum Increases Glomerular HO1 Expression three.two. HPXDeficientwhether HPX deficient serum increases glomerular HO1 expression, isolateddetermine regardless of whether HPX deficient serum increases glomerular HO1 expression, glomeruli had been incubated ToTo decide no matter whether HPXin serumfree media or media supplemented with 2.5 deficient serum increases glomerular HO1 expression, (v/v) HPXserum had been incubated in serumfree absence of exogenous heme (hemin). As isolated glomeruli for 18 h inside the presence or media oror media supplemented with 2.5 isolated glomeruli had been incubated in serumfree media media supplemented with 2.5 shown in elevated (v/v) HPXFigure two, HO1 protein expression wasabsence of in glomeruli incubated with (v/v) HPX erum for18 hh within the presence or serum for 18 in t.
