Anti gst

Proteins were separated on a SDS-PAGE gel and transferred to a nitrocellulose membrane. After transfer, the membrane was blocked in TBSTM (Tris-buffered saline, 0.5% Tween20, 5% Milk) for 30 minutes. Probing with the indicated primary antibodies in blocking solution was for 2 hours at room temperature (RT) or overnight at 4 °C. Donkey anti-mouse (IRDye 680LT) or anti-rabbit (IRDye 800CW) secondary antibodies (LiCOR) were added for 1 hour at RT. Blots were imaged using the LiCOR Odyssey system. Primary antibodies used for western blotting were anti-YY1 (H-10), anti-Cyclin B1, and anti-GAPDH, anti-GST, anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Aurora A (D3E4Q) (Cell Signaling Technology, Danvers, MA), and anti-Flag (Sigma). Anti-HpTGEKP antibody was previously described^{50 (link)}. The rabbit polyclonal anti-YY1pS365 was generated by New England Peptide Inc. (Gardner, MA) using a synthesised phospho-peptide corresponding to YY1 amino acids 360–369 (Ac-CGKRF(p)SLDF-amide).

Proteins were revealed using the following primary antibodies: rabbit polyclonal anti-GST (Santa Cruz Biotechnology), rabbit polyclonal anti-PP5 (Abcam) and anti-rabbit HRP-conjugated secondary antibodies (Pierce). Cell extracts were separated in 4%–12% Bis-Tris NuPAGE gels (Life) and electro-blotted onto PVDF membranes (Pierce).

To prepare glutathione S-transferase (GST)-tagged TSF for the in vitro pull-down assays, the full-length CDS of TSF was cloned into the pGEX-5X-1 vector and introduced into E. coli BL21 cells. GST only or GST-tagged TSF was expressed in E. coli BL21 cells at 28°C with 0.15 mM IPTG. After the protein extracts were sonicated in GST lysis buffer (50 mM Tris–HCl pH 7.5, 0.1 M NaCl, 0.05% Tween-20, 1 mM EDTA pH 8.0, 1 mM PMSF, and protease inhibitor cocktail), the cell lysates were incubated in a glutathione-Sepharose 4B (GE healthcare) slurry for 1 h at 4°C and washed three times with the same lysis buffer.
For the in vitro pull-down experiment, purified His-tagged FRK6 and His-tagged FRK7 were incubated with equal amounts of GST only or GST-fused TSF immobilized on glutathione-Sepharose 4B beads for 1 h at 4°C. After the binding reaction, the beads were washed four times with GST lysis buffer. Proteins bound to beads were dissociated by adding SDS–PAGE sample buffer and loaded onto a 15% SDS–PAGE gel. Immunoblotting was performed using anti-His (Santa Cruz) or anti-GST (Santa Cruz) primary antibodies and goat anti-rabbit IgG secondary antibodies. The bands were visualized by applying Enhanced Chemiluminescence solution (AbClon).

All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously^{26 (link)}. Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

All Ephexin4 mutants were generated by a polymerase chain reaction (PCR)-based strategy from the murine Ephexin4 cDNA (NM_001112744). Specifically, two point-mutated Ephexin4 (P271A and E295A) were generated using site-directed mutagenesis. GST-Elmo2^1-360 was constructed by inserting the N-terminal part of the murine Elmo2 cDNA (NM_080287) into the pGEX-4T-2 vector. GFP-RhoG was used in the previous study [19 (link)]. The antibodies used in this study were anti-FLAG (Sigma, St. Louis, MO, USA, M2), anti-GFP (Santa Cruz Biotechnology, Dallas, TX, USA, FL), anti-GST (Santa Cruz Biotechnology, B-14), and anti-RhoG (Santa Cruz Biotechnology, 1F3 B3 E5).