Anti flag primary antibody

15 µl of each IP sample was loaded into 4–15% gradient SDS-PAGE gels (Bio-Rad). The gels were run at 25 mA for 2 hr, and blotted to PVDF membrane. After 1 hr blocking in Li-Cor blocking buffer, the membrane was incubated with anti-FLAG primary antibody (SIGMA, F3165) for 1 hr, anti-Ssa1/2 (gift from V. Denic) or anti-HIS antibody (all 1:1000 dilutions). The membranes were washed three times with TBST. The proteins were probed by anti-mouse-800 IgG (Li-Cor, 926–32352, 1:10000 dilution). The fluorescent signal scanned with the Li-Cor/Odyssey system. For the heat shock time course, cells expressing Hsf1-3xFLAG-V5 were grown to OD₆₀₀0.8 in 25 ml YPD at 25°C. At time t = 0, 25 ml of 53°C media was added to instantly bring the culture to 39°C and then the culture was incubated with shaking at 39°C. 5 ml samples were collected at each time point by centrifugation. Pellets were boiled with 2X SDS loading buffer for 10 min. Total protein concentration was measured by NanoDrop and an equal amount of each sample was loaded into 7.5% SDS-PAGE gel and otherwise processed as above.

Dissected brains were embedded in Tissue-Tek (Sakura, Japan) and freshly frozen in dry-ice ethanol. Cryo-sections at a thickness of 10 µm were collected on PLL-coated slide glasses, fixed in 4% PFA for 10 minutes at a room temperature, and permeabilized in 100% MetOH at −20 °C for 5 minutes. After rehydration in PBS, the sections were incubated with anti-FLAG primary antibody (#F1804, Sigma-Aldrich) and were subsequently incubated with anti-mouse Cy3-conjugated secondary antibodies (Millipore, AP124C). Fluorescent images were obtained using an epifluorescence microscope (BX51, Olympus) equipped with a CCD camera (DP70).

After induction in galactose media overnight, cells were lysed using glass beads in lysis buffer (100 mM Tris pH 7.5; 200 mM NaCl; 1 mM EDTA; 5% glycerol, 1 mM Dithiothreitol (DTT) 4 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail). Equal amount of proteins were spotted on a nitrocellulose membrane. Membranes were blocked for 30 min in PBS-0.05%Tween at room temperature were and then processed for immunoblot. Membranes were probed with anti-FLAG primary antibody (Sigma F3040, 1:5000 dilution) overnight at 4°C and subsequently with a secondary anti-mouse fluorescent antibody (Thermo Alexa 555 #A21424, 1:5000 dilution) for 1h at room temperature and imaged using a Bio-Rad ChemiDoc MP imaging system. Membranes were then stripped using the Gene Bio-Application stripping buffer and reprobed with an anti-Pgk1 primary antibody (Thermo 22C5D8) using the same secondary antibody. In
Figure 3, for each individual antibody, both membranes were imaged simultaneously to allow direct comparison of fluorescent signal. Densitometric analysis was performed using Image J.

Cell surface expression was determined by flow cytometry to the N-terminal Flag tag on the WT VIP2R(1-23-Flag-VIP2R(24–438)) and its mutants transiently expressed in CHO-K1 cells. All the mutant constructs were modified by single-point mutation in the setting of the WT construct. Briefly, approximately 3 × 10⁵ cells were blocked with PBS containing 5% BSA (w/v) at RT for 15 min, and incubated with 1:300 anti-Flag primary antibody (diluted with PBS containing 5% BSA, Sigma-Aldrich) at RT for 1 h. The cells were then washed three times with PBS containing 1% BSA (w/v) followed by 1 h incubation with 1:1000 anti-mouse Alexa Fluor 488 conjugated secondary antibody (diluted with PBS containing 5% BSA, Invitrogen) at 4 °C in the dark. After washing three times, cells were re-suspended in 200 μL PBS containing 1% BSA for detection by Flow Cytometer (BD Biosciences) utilizing laser excitation and emission wavelengths of 488 and 530 nm, respectively. For each sample, 10,000 cellular events were collected, and the total fluorescence intensity of positive expression cell population was calculated by NovoExpress 1.2.1. Data were normalized to the WT receptor.

The 3 × Flag fused MoFBP1 or MoPCK1 construct was constructed. Both constructs were transformed into the wild type or ΔMoubp14 mutant, respectively. Subsequent transformants were performed with Western blot analysis with an anti-Flag primary antibody (1:5000, Sigma, United States) and an anti-rabbit horseradish peroxidase secondary antibody (1:10000, Sigma, United States). Results were visualized with the ECL detection system (Amersham Biosciences, United Kingdom). To test catabolite degradation of MoFbp1 and MoPck1, the wild type and the ΔMoubp14 mutant strains expressing Fbp1:3 × Flag or Pck1: 3 × Flag were incubated firstly in CM medium for 42 h, and then transferred the mycelia into the liquid medium with NaAc as the sole carbon source for 12 h, and then added glucose into the medium for incubating for 2 h. Total ubiquitination levels of the wild type, the ΔMoubp14 mutant and the complementation strain cUBP14 were detected by an anti-ubiquitin antibody (Abcam, United Kingdom).

HEK293 cells were transiently transfected with N-terminal Flag-tagged wild type CRF2R or mutants in 24-well plates. 48 hours after transfection, the cells were fixed with 4% (w/v) formaldehyde for 10 min followed by incubation in blocking solution (5% BSA in DPBS) for 1 hour at room temperature. The cells were incubated with anti-FLAG primary antibody (Sigma Aldrich, Cat# F1804, 1:1000) followed by incubation with secondary anti-mouse antibody (Thermo Fisher, Cat# A-21235, 1:5000) conjugated to horseradish peroxide. The tetramethyl benzidine (TMB/E) solution was added and the reaction was terminated by adding 0.25 M HCl solution. The absorbance at 450 nm was measured using the TECAN luminescence counter (Infinite M200 Pro NanoQuant) to characterize the cell surface expression level of each receptor. The expression levels of the mutants were normalized to that of the WT CRF2R. Data are shown as the mean ± SEM. Data are from three independent experiments (n = 3).