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. 2022 Nov 17;298(12):102713. doi: 10.1016/j.jbc.2022.102713

Figure 1.

Figure 1

LH3 deficiency in PFHR9 cells impairs collagen α1α1α2(IV) secretion.A, semiquantitative Western blots showing successful ablation of LH3 in PFHR9 cells following CRISPR/Cas9-mediated LH3 inactivation (LH3 KO PFHR9 cells). β-tubulin was used as a loading control. B, semiquantitative Western blots and quantification of COL4A1 and COL4A2 levels in the conditioned cell culture medium showing reduced secretion of COL4A1 and COL4A2 in LH3 KO PFHR9 cells (KO) compared with their WT controls. Laminin α/β/γ was used as a loading control. C, semiquantitative Western blots and quantification of COL4A1 and COL4A2 levels in cell lysates showing intracellular COL4A1 accumulation in LH3 KO PFHR9 cells. β-tubulin was used as a loading control. COL4 indicates control-purified collagen α1α1α2(IV). The quantification data in B and C are shown as fold relative to WT levels and presented as means ± SD with individual data points representing independent preparations of culture medium and cell lysate and (n = 3). D, representative SDS-PAGE gel (top) and quantification (bottom) of time-dependent extracellular collagen α1α1α2(IV) levels in WT PFHR9 cells measured by AHA-Alexa Fluor pulse-chase labeling. “ctrl” indicates purified AHA-incorporated collagen α1α1α2(IV). The quantification of AHA-incorporated collagen α1α1α2(IV) was set to 1.0 at 480 min (n = 5). E, representative SDS-PAGE gel (top) and quantification (bottom) of time-dependent extracellular collagen α1α1α2(IV) levels for LH3 KO PFHR9 cells measured by AHA-Alexa Fluor pulse-chase labeling, demonstrating significantly impaired collagen α1α1α2(IV) secretion in LH3 KO PFHR9 cells compared with their WT AHA-incorporated collagen α1α1α2(IV) controls at 8 h (n = 5). Source data files for the figure: The uncropped images of Western blots used in the figure are presented in Fig. S2; generation of the purified AHA-incorporated collagen α1α1α2(IV) used as a control in the figure is presented in Fig. S3; and the value of AHA-incorporated collagen α1α1α2(IV) at each time points is shown in Table S1. AHA, l-azidohomoalanine; LH3, lysyl hydroxylase 3.