TITLE: mCherry-YIPF4 Immunoprecipitation V2
AUTHORS: Kelsey Hickey, Sharan Sharan Swarup, Harper JW

[DESCRIPTION]
This protocol is for immunoprecipitating an mcherry tagged Golgi protein- YIPF4, and probing for ATG8 interactions. 

[STEPS]
SECTION: HEK293 YIPF4 KO Creation
1. Maintain HEK293 cells in Dulbecco’ Modifies Eagles Medium (DMEM) with 10% fetal bovine serum and optional 1x penicillin-streptomycin. 
SECTION: HEK293 YIPF4 KO Creation
1. Individual clones were subjected to immunoblotting with anti-YIPF4 (Sino Biological 202844-T46), clones lacking the relevant protein were selected for further analysis by Sanger sequencing of the edited alleles.
SECTION: HEK293 YIPF3/YIPF4 Double KO Creation
2. For YIPF3/YIPF4 double knock-out, the following sgRNA sequence within the YIPF3 gene was designed and ordered (5’  CCATTTCGGGCGCCGCCCGC  3' ), and cloned into a pX459 vector to create pX459-gRNA-YIPF3-KO.  
Sequence validate by Sanger sequencing.


SECTION: HEK293 YIPF3/YIPF4 Double KO Creation
2.1. Transfect YIPF4 KO HEK293 cells with the pX459-gRNA-YIPF3-KO using Lipofectamine 3000, and select with 1.2 µg/mL of puromycin for 24-48 hours. Select monoclonal cells by limiting dilution or by cell sorting (SONY SH800S sorter) in 96 well plates. 
SECTION: HEK293 YIPF3/YIPF4 Double KO Creation
2.2. Individual clones were subjected to immunoblotting with anti-YIPF4 (Sino Biological 202844-T46) and anti-YIPF3 (Invitrogen PA566621), clones lacking the relevant protein were selected for further analysis by Sanger sequencing of the edited alleles.
3. 
SECTION: HEK293 YIPF3/YIPF4 double KO creation
2. For YIPF3/YIPF4 double knock-out, the following sgRNA sequence was designed and ordered within YIPF3  (5’ GCGTACAAGCTGAAGGCCC 3' ), and cloned into a pX459 vector to create pX459-gRNA-YIPF3-KO.  
SECTION: HEK293 YIPF4 KO Creation
1.1. For YIPF4 knock-out, the following sgRNA sequences were designed and ordered within YIPF4 (5’  ATCTCGCGGCGACTCCCAAC 3' / 5’ CGGCCTATGCCCCCACTAAC 3' ), and cloned into a pX459 vector to create pX459-gRNA-YIPF4-KO.  
SECTION: HEK293 YIPF4 KO Creation
1.2. Transfect HEK293 cells with the pX459-gRNA-YIPF4-KO with Lipofectamine 3000, and select with 1.2 µg/mL of puromycin for 24-48 hours. Select monoclonal cells by limiting dilution or by cell sorting (SONY SH800S sorter) in 96 well plates.
SECTION: HEK293 YIPF4 KO Creation
1.3. Individual clones were subjected to immunoblotting with anti-YIPF4 (Sino Biological 202844-T46), and clones lacking the relevant protein were selected for further analysis by Sanger sequencing of the edited alleles.
SECTION: HEK293 YIPF3/YIPF4 double KO creation
2.1. Transfect YIPF4 KO HEK293 cells with the pX459-gRNA-YIPF3-KO with Lipofectamine 3000, and select with 1.2 µg/mL of puromycin for 24-48 hours. Select monoclonal cells by limiting dilution or by cell sorting (SONY SH800S sorter) in 96 well plates.
SECTION: HEK293 YIPF3/YIPF4 double KO creation
2.2. Individual clones were subjected to immunoblotting with anti-YIPF3 (Invitrogen PA566621), and clones lacking the relevant protein were selected for further analysis by Sanger sequencing of the edited alleles.
SECTION: HEK293 YIPF3/YIPF4 double KO creation
2.3. 
SECTION: IP
3. DKO (YIPF3/4) HEK293 cells were reconstituted with mCherry-YIPF4 (WT or LIR mutant) and GFP-YIPF3 (WT or LIR mutant) constructs and sorted for equal expression levels. 
Immunofluorescence was used to confirm proper localization of both YIPF3 and YIPF4
SECTION: IP
3.1. cells were plated on 10cm plates and grown to 70% confluency. Cells were left untreated or starved using AA withdrawal for 2 hours in the presence of BafA (100nm)
SECTION: IP
3.2. Cells were washed 2x with cold PBS then lysed in 0.8mL of NP-40 lysis buffer (100mM TRIS pH7.4, 150mM KCL, 0.1% NP-40, 0.5mM EDTA, 1x HALT (Roche) protease inhibitors, phos-STOP tabs)
SECTION: IP
3.3. 1.5mg of protein from each sample was added to 15ul of washed RFP-TRAP beads and incubated for 2 hours while rotating at 4 degrees. Beads were washed 3 times with lysis buffer and eluted in 1x LDS loading dye at 94 degrees for 5 minutes
SECTION: IP
3.4. 
SECTION: IP
3.5.