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. 2024 Oct 31;13:RP96942. doi: 10.7554/eLife.96942

Figure 2. Expression of YFP-FMNL1β S1086 variants in FMNL1-silenced cells.

(A) C3 control clone was untransfected (Control YFP-) (first row) or transfected with FMNL1-interfering (shFMNL1-HA-YFP) (second row), or FMNL1-interfering expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third row), YFP-FMNL1βS1086A (fourth row), or YFP-FMNL1βS1086D (fifth row) constructs. Subsequently, cells were challenged with CMAC-labeled SEE-pulsed Raji cells (blue) for 1 hr, fixed, stained with anti-FMNL1, and imaged by epifluorescence microscopy. Representative maximum intensity projection (MIP) images of merged transmittance (TRANS), CMAC (blue), YFP (yellow), and anti-FMNL1 (red) channels are indicated for the different cell groups. (B) Cell lysates corresponding to the indicated cell groups were analyzed by western blot (WB) developed with anti-FMNL1 antibody (two different expositions). The lower bar graph depicts the WB quantification showing the endogenous FMNL1 expression in the different cell groups relative to control untransfected (YFP-) cells. Results are representative of data from several independent experiments (n=3) with similar results.

Figure 2—source data 1. Original, uncropped WB corresponding to panel B in Figure 2 in PDF format.
Figure 2—source data 2. Original, uncropped WB corresponding to panel B in Figure 2 in TIF format.

Figure 2.

Figure 2—figure supplement 1. Expression of YFP-FMNL1β constructs and correlation between YFP-FMNL1β variants expression and the anti-FMNL1 antibody signal.

Figure 2—figure supplement 1.

(A) Scatter plots of the mean fluorescence intensity (MFI) values of YFP or YFP-FMNL1 variants and the anti-FMNL1 signal in cells transfected with FMNL1-interfering (shFMNL1-HA-YFP), YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT), YFP-FMNL1βS1086A, or YFP-FMNL1βS1086D constructs are depicted. The trend line (linear regression) is displayed in red color in FMNL1β constructions. (B) Box plot representing the different anti-FMNL1 MFI signals in the control and the different transfected cell groups. This figure is related to Figure 2. ***, p≤0.05. Results and ANOVA are representative of the data from several independent experiments (n=3) with similar results.