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. 2020 Feb 7;295(15):5036–5050. doi: 10.1074/jbc.RA119.011992

Figure 3.

Figure 3.

Assessment of physical interaction between cortical actin and the LYVE-1 cytoplasmic tail in HDLECs by co-immunoprecipitation. A and B, pulldowns of LYVE-1:actin complexes using either biotinylated LYVE-1 (bLYVE-1) (A) or bPhal (B) analyzed by SDS-PAGE and blotting with appropriate β-actin and LYVE-1 Abs, respectively (see “Experimental procedures”). Control immunoblots are also shown for matched loadings of HDLEC whole-cell lysates (WCL) dissolved directly in SDS-PAGE sample buffer (SB) and the soluble fraction from HDLECs lysed with the ASB used for subsequent immunoprecipitation (i.p.) (S/N, supernatant). Note that all three lanes are from the same corresponding gels in each case and spliced as shown. Bar charts show quantification using IR dye–conjugated secondary Abs and LI-COR® imaging (mean ± S.E. (error bars) (n = 5 in A; unpaired Student's t test, p = 0.0001; n = 3 in B, unpaired Student's t test, p = 0.0132). C, corresponding pulldowns of LYVE-1:actin complexes from HDLECs transfected with either FL LYVE-1 or LYVE-1 Δ263 or Δ259 cytoplasmic tail truncation mutants and LI-COR imaging (mean ± S.E., n = 4 for LYVE-1 Δ263, p = 0.0035, unpaired t test; mean ± S.E., n = 3 for LYVE-1 Δ259, p = 0.0002, unpaired t test). Note that all four lanes are from the same corresponding gels in each case and spliced as shown. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.