Immobilon fl transfer membrane

Fly tissues were homogenized in SDS sample buffer with a pellet pestle (Kimble Chase, Vineland, NJ, USA), and the proteins were fractionated by SDS-PAGE. Proteins from the gels were then transferred onto Immobilon-FL transfer membranes (Millipore, Danvers, MA, USA) in Tris-glycine buffer. The blots were probed with anti-FLAG (Mouse; Sigma, 1:2000 dilution), anti-tubulin (mouse; Developmental Studies Hybridoma Bank, Iowa City, IA, USA, 1:5000 dilution), or anti-GFP (rabbit; Torrey pines Biolabs Inc., San Diego, CA, USA, 1:2000 dilution) as primary antibodies, followed by IRDye 800 goat anti-rabbit IgG (LI-COR) or IRDye 680 goat anti-mouse IgG as secondary antibodies. Signals were detected using an Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA).
To evaluate the phosphorylation levels of the PINK1 and Parkin proteins in the absence of phosphorylation site-specific antibody, 6% polyacrylamide gels containing 50 μM phostag acrylamide (Wako Chemicals, Osaka, Japan) and 100 μM MnCl₂ were used. After electrophoresis, phostag acrylamide gels were washed in general transfer buffer containing 0.02% SDS and 2 mM EDTA and then replaced with general transfer buffer lacking EDTA. After completely washing out manganese ions, the proteins were transferred onto Immobilon-FL transfer membranes (Millipore) and immunoblotted using a standard protocol.

The ganglia were mechanically dissociated using Precellys 24 Bertin technologies into a RIPA lysis buffer (Thermofisher, Rockford, IL, USA). After tissue lysis, homogenates were centrifuged (4 °C, 10,000 rpm) for 10 min. Pellets were resuspended in a Laemmli buffer (Bio-Rad). After protein quantification, 50 µg of protein was used to perform electrophoresis (Tris-HCl PAGE-Gel (Bio-Rad laboratories Inc., Hercules, CA, USA). The transfer of proteins was performed using an activated-methanol membrane (0.35 A) (Merck Millipore, Tullagreen, Carrigtwohill, Co, Cork, Ireland, Immobilon FL Transfer Membrane) at 4 °C for 90 min. Blocking and antibody incubation were performed using the IBindTM Flex Western System SLF2000 (Invitrogen). The intensity of each band was determined by pixel density integration using LI-COR^® Odyssey Western Blotting Kits. Antibodies used are presented in Table 1.

Harvested E. coli cells were treated with lysozyme, and whole-cell extracts were prepared by sonication. Total cell proteins were fractionated using Tricine-SDS-PAGE on 15% gels [51 (link)] and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-FL transfer membrane; Merck, Darmstadt, Germany). The proteins Rsd, RMF, YqjD, RpoA, and RplB were detected on the membranes using rabbit polyclonal antibodies against Rsd, RMF, YqjD, RpoA, and RplB, respectively. Immunostained membranes with ECL substrate (Cytiva, Tokyo, Japan) were scanned using ImageQuant LAS 500 (Cytiva, Tokyo, Japan). The density of each band on the membranes was quantified using ImageJ software (https://imagej.nih.gov/ij/index.html (accessed on 24 August 2022)). The linearity of the quantification was confirmed through several experiments with different amounts of sample solution loaded on an electrophoresis gel.