Tbc1d24

Cells were fixed with 4% PFA at RT for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min. For blocking, cells were incubated with 2% FBS in PBS for 1 hr at RT. Cells were then incubated at 4°C overnight with the following primary antibodies: Tbc1d24 (ProteinTech, 25254-1-AP, 1:200, LSBio, LS-C679739, 1:200, Washington, US), Vimentin (Santa Cruz, sc-6260, 1:100, Texas, US), Impdh (Santa Cruz, sc-36-5171, 1:100, ProteinTech, 12948-1-AP, 1:100), Cpts1 (ProteinTech, 15914-1-AP, 1:200 and LSBio, LS-C197001, 1:100). After washing with PBS, cells were incubated with anti-mouse IgG conjugated with Alexa Fluor 546 (Invitrogen, A11030, 1:1000) or anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen, A11008, 1:1000) for 1 hr at RT. Prolong Gold Antifade mountant with DAPI (Invitrogen) was used for mounting and nuclear staining. Cellular images were taken with a confocal microscope (Leica, TCP SP8, Wetzlar, Germany) or a super-resolution fluorescence microscope (GE healthcare, DeltaVision Elite). The length (μm) of cytoophidia was measured using Fiji [39 (link)].

Cell lysates were prepared using RIPA lysis buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. The lysates were centrifuged at 15,000 rpm for 10 min. The lysates were mixed with Laemmli sample buffer and boiled at 98°C for 2 min. The protein samples were run on SDS-PAGE along with protein markers for SDS-PAGE (Nacalai Tesque, 02525–35, Kyoto, Japan) and blotted onto a PVDF membrane (GE healthcare, Illinois, US). The following primary antibodies were used: Tbc1d24 (ProteinTech, 1:1000, Illinois, US; LSBio, 1:1000, Illinois, US), p21 Waf1 (Abcam, ab109199, 1:1000, Cambridge, UK), p27 Kip1 (Cell Signaling Technology, #3698, 1:1000, Massachusetts, US), Impdh (ProteinTech, 12948-1-AP, 1:100), Cpts1 (ProteinTech, 15914-1-AP, 1:100), Arf6 (Cell Biolabs, STA-407-6, 1:500, California, USA) and beta-actin (Cell Signaling Technology, #5174, 1:4000). HRP-conjugated anti-mouse IgG (Thermo Fisher Scientific, 32430) and anti-rabbit IgG (Thermo Fisher Scientific, 32460) were used as secondary antibodies at 1:1000 dilution. For detection of immunoreactive bands, Chemi-Lumi One L or Chemi-Lumi One Ultra (Nacalai Tesque) were used. Uncropped blots were shown in S1 Raw images. Densitometric analyses for blotting data were performed using Fiji [39 (link)].