Figure S3. Analysis of the OPTN – PEX14... | Rockefeller University Press

Figure S3.

$Analysis of the OPTN–PEX14 interaction using GFP trapping. (A) Immunoblot analysis of input and GFP-Trap pull-down fractions from Flp-In T-REx 293 cells expressing the specified proteins. Representative images are shown. Specific protein bands and their degradation products are marked by arrows and arrowheads, respectively. Molecular mass markers (in kDa) are indicated on the left. (B) Densitometry quantifications of the relative ratios (RRs) of PEX14/GFP and PEX14/OPTN-GFP retained on the GFP-Trap affinity matrix. The total signal intensities of the GFP proteins and PEX14 were both standardized to 100%. The bars represent the mean of two biological replicates. T, transient transfection; F, Flp-In integration; L, lentiviral transduction. (C and D) Relative expression of GFP-tagged OPTN compared with endogenous OPTN in lentiviral-transduced Flp-In T-REx 293 cells. (C) Total cell lysates from lentiviral (L-) transduced Flp-In T-REx 293 cells, which constitutively express either GFP or OPTN-GFP, were subjected to SDS-PAGE followed by IB using antibodies targeting OPTN and GAPDH. A representative image is shown, with specific protein bands indicated by arrows. Note that the 66 kDa band in the OPTN-GFP lane may represent either a degradation product of OPTN-GFP or endogenous OPTN stabilized by overexpression of OPTN-GFP. Molecular mass markers (in kDa) are indicated on the left. (D) Densitometry quantifications of the relative normalized amounts (R-NA) of OPTN. The expression levels were normalized to GAPDH, with the amount of OPTN detected in the GFP cells serving as the reference. The amount in the L-OPTN-GFP cells represents the sum of the 66- and 95-kDa bands. Bars represent the mean ± SD of four biological replicates (****, P < 0.0001). Source data are available for this figure: SourceData FS3.$

Analysis of the OPTN–PEX14 interaction using GFP trapping. (A) Immunoblot analysis of input and GFP-Trap pull-down fractions from Flp-In T-REx 293 cells expressing the specified proteins. Representative images are shown. Specific protein bands and their degradation products are marked by arrows and arrowheads, respectively. Molecular mass markers (in kDa) are indicated on the left. (B) Densitometry quantifications of the relative ratios (RRs) of PEX14/GFP and PEX14/OPTN-GFP retained on the GFP-Trap affinity matrix. The total signal intensities of the GFP proteins and PEX14 were both standardized to 100%. The bars represent the mean of two biological replicates. T, transient transfection; F, Flp-In integration; L, lentiviral transduction. (C and D) Relative expression of GFP-tagged OPTN compared with endogenous OPTN in lentiviral-transduced Flp-In T-REx 293 cells. (C) Total cell lysates from lentiviral (L-) transduced Flp-In T-REx 293 cells, which constitutively express either GFP or OPTN-GFP, were subjected to SDS-PAGE followed by IB using antibodies targeting OPTN and GAPDH. A representative image is shown, with specific protein bands indicated by arrows. Note that the 66 kDa band in the OPTN-GFP lane may represent either a degradation product of OPTN-GFP or endogenous OPTN stabilized by overexpression of OPTN-GFP. Molecular mass markers (in kDa) are indicated on the left. (D) Densitometry quantifications of the relative normalized amounts (R-NA) of OPTN. The expression levels were normalized to GAPDH, with the amount of OPTN detected in the GFP cells serving as the reference. The amount in the L-OPTN-GFP cells represents the sum of the 66- and 95-kDa bands. Bars represent the mean ± SD of four biological replicates (****, P < 0.0001). Source data are available for this figure: SourceData FS3.