HS1 silencing affects F-actin polymerization, distribution, and activation of cytoskeleton components. (A) Relative expression of HS1 evaluated by real-time PCR in 3 stable cell lines obtained from MEC1 cell line, either expressing GFP only (CNTR) or after HS1 silencing (HS1 KD1 and KD2). (B) Western blot analysis for HS1 on silenced cell lines (CNTR, KD1, and KD2); β-actin is used as control. Graph represents the densitometric analysis to quantify the protein expression levels. Because the silencing was more effective in HS1 KD1 cells in further experiments, we used only this cell line that is from now on defined as HS1 KD. (C) Immunoprecipitation experiments for total phosphotyrosine on CNTR and HS1 KD cell protein lysates; the gel is blue-Coomassie stained, and bands 1, 2, 3, and 4 were trypsin digested and analyzed by high-resolution mass spectrometry (see “Results” and Table 1). (D) Confocal analysis of CNTR and HS1 KD cells with anti-actin and anti–myosin heavy chain 9 (MYH9) primary antibodies detected with Alexa Fluor 546 (yellow)– and 633 (red)–conjugated secondary antibodies. White arrows indicate characteristic CNTR cell protrusions and HS1 KD dotted actin accumulation, the overlap between Alexa Fluor 546 and 633 is orange. Images were acquired with a laser scanning confocal microscope (Leica SPS) with the use of an inverted 63× oil objective equipped with a resonant scanner. (E) Total internal reflection fluorescence acquisition after Phalloidin Alexa Fluor 633 staining on CNTR cells and HS1 KD adhered on poli-L-Ornithine coating, arrows indicate the different F-actin distribution. Images were acquired with a TIRF microscope (Leica AM) with the use of a 60× oil objective equipped with an Andor ENCCD camera. (F) F-actin polymerization is represented as the ratio of the flow cytometric mean fluorescence intensity (MFI) values after and before IgM stimulation in WT and HS1^−/− mice B splenocytes (6 animals/group).